JP2021073198A - Tolerogenic synthetic nanocarriers and therapeutic macromolecules for reduced or enhanced pharmacodynamic effects - Google Patents

Abstract

To provide compositions and methods that provide pharmacodynamic effects specific to therapeutic macromolecules.SOLUTION: The effects may result from reduced doses of therapeutic macromolecules in combination with immunosuppressant doses. The effects may also be enhanced with such compositions.SELECTED DRAWING: None

Description

Related Applications This application is under Article 119 of the US Patent Law, US Provisional Application No. 61/819517 filed on May 3, 2013, No. 61/881851 filed on September 24, 2013, 2013 9 No. 61/881913 filed on March 24, No. 61/881921 filed on September 24, 2013, No. 61/907177 filed on November 21, 2013, filed on March 5, 2014. It claims the interests of No. 61/948313 and No. 61/948384 filed March 5, 2014, the entire contents of each of which are incorporated herein by reference.

INDUSTRIAL APPLICABILITY The present invention relates to immunosuppressive drug doses, methods that are attached to synthetic nanocarriers in some embodiments, administered in combination with therapeutic macromolecules, and related. The compositions and methods allow for efficient pharmacodynamic effects specific to therapeutic macromolecules. Therefore, using the provided compositions and methods, even reduced doses of therapeutic macromolecules can cause a pharmacodynamic response in the subject. The compositions and methods provided herein can also be administered in combination repeatedly to produce the desired pharmacodynamic and immunological effects.

Background of the Invention A therapeutic treatment, such as protein or enzyme replacement therapy, often results in an undesired immune response to a particular therapeutic agent. In such cases, cells of the immune system recognize the therapeutic agent as a foreign body and attempt to neutralize or destroy it, just as they would destroy an infectious organism such as a bacterium or virus. Such an undesired immune response may neutralize the effectiveness of the therapeutic procedure or cause a hypersensitivity response to the therapeutic agent. These undesired responses can be reduced through the use of immunosuppressive drugs. However, conventional immunosuppressants are broad-acting, and the use of broad-acting immunosuppressants is associated with the risk of serious side effects such as tumors, infections, nephrotoxicity and metabolic disorders. Therefore, new treatments are beneficial.

Overview of the Invention In one aspect, an immunosuppressive agent dose, wherein in some embodiments, an immunosuppressive agent dose is attached to a synthetic nanocarrier, and an immunosuppressive agent dose and a Therapeutic Polymer. Methods are provided that include co-administration of a reduced pharmacodynamically effective dose to a subject. In one aspect, concomitant administration has reduced pharmacodynamics of the therapeutic polymer when co-administered with an immunosuppressive drug dose compared to administration of a therapeutic polymer when not co-administered with an immunosuppressive drug dose. Each is in the presence of an anti-therapeutic high molecular weight antibody response, according to protocols that have been demonstrated to produce pharmacodynamic effects at effective doses. In another aspect of any one of the methods provided, a reduced pharmacodynamically effective dose of the therapeutic polymer is administered in the presence of (A) an anti-therapeutic polymer antibody response and (B). Less than the pharmacodynamically effective dose of therapeutic macromolecules, not co-administered with the immunosuppressant dose.

In another aspect, providing an immunosuppressive drug dose, wherein in some embodiments the immunosuppressive drug dose is attached to a synthetic nanocarrier, and the pharmacodynamics of the immunosuppressive drug dose and the therapeutic polymer. Methods are provided that include co-administration with an effective dose. In one aspect, concomitant administration provides a pharmacodynamic effect of the therapeutic macromolecule when co-administered with an immunosuppressive drug dose as compared to administration of a therapeutic macromolecule when not co-administered with an immunosuppressive drug dose. Each is in the presence of an anti-therapeutic polymeric antibody response, according to protocols that have been proven to improve.

In another aspect, providing an immunosuppressive drug dose, wherein in some embodiments the immunosuppressive drug dose is attached to a synthetic nanocarrier, the immunosuppressive drug dose and the pharmacodynamic efficacy of the therapeutic polymer. Methods are provided that include co-administering the dose with and recording the improved pharmacodynamic effect following the concomitant administration.

In another aspect, providing a therapeutic polymer that causes or is expected to cause an anti-therapeutic macromolecular antibody upon repeated dosing in one or more subjects, and an immunosuppressive drug dose, A method comprising providing, where an immunosuppressive agent dose is attached to a synthetic nanocarrier. In some embodiments, the method comprises administering the therapeutic macromolecule at the same or less dose to the subject repeatedly in combination with an immunosuppressant dose. In some embodiments, the combination administration follows a protocol that has been demonstrated to result in maintenance of the pharmacodynamic effect of the therapeutic macromolecule over two or three or more doses of the therapeutic macromolecule to the subject.

In one aspect of any one of the provided methods, the method further comprises determining a protocol. In another aspect of any one of the provided methods, the method further comprises determining a pharmacodynamically effective dose, such as a reduced or improved pharmacodynamically effective dose. In another aspect of any one of the provided methods, the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration. In another aspect of any one of the provided methods, the combination administration is repeated once or more than once. In another aspect of any one of the methods provided, administration is by intravenous, intraperitoneal or subcutaneous administration. In another aspect of any one of the provided methods, the subject is at risk of an anti-therapeutic macromolecular antibody response. In another aspect of any one of the provided methods, the subject is expected to experience an anti-therapeutic macromolecular response.

In another aspect, a composition or composition comprising an immunosuppressant dose, wherein in some embodiments the immunosuppressant is attached to a synthetic nanocarrier, and a reduced pharmacodynamically effective dose of the Therapeutic macromolecule. A kit is provided.
In another aspect, use in any one of the methods provided herein, combined with an immunosuppressant dose, wherein in some embodiments the immunosuppressant is attached to a synthetic nanocarrier. Compositions or kits containing reduced pharmacodynamically effective doses of therapeutic macromolecules are provided.

In any one aspect of the provided composition or kit, the composition or kit is for use in any one of the methods provided herein. In any one aspect of the provided composition or kit, the composition or kit further comprises a pharmaceutically acceptable carrier.
In any one aspect of the provided method or composition or kit, the therapeutic macromolecule is not attached to the synthetic nanocarrier. In any one other aspect of the provided method or composition or kit, the therapeutic macromolecule is attached to a synthetic nanocarrier. In any one other aspect of the provided method or composition or kit, the synthetic nanocarrier does not contain the APC presentable antigen of the therapeutic macromolecule.

In any one aspect of the provided composition or kit, the immunosuppressant dose and the therapeutic macromolecule are contained in the container, respectively. In any one other aspect of the provided composition or kit, the immunosuppressant dose and therapeutic macromolecule are contained in separate containers. In any one other aspect of the provided composition or kit, the immunosuppressant dose and therapeutic macromolecule are contained in the same container.

In any one aspect of the methods or compositions or kits provided, reduced pharmacodynamically effective doses of therapeutic macromolecules are administered in the presence of (A) anti-therapeutic macromolecular antibody response. And (B) at least 30% less than the pharmacodynamically effective dose of the therapeutic macromolecule that is not co-administered with the immunosuppressant dose. In any one other aspect of the method or composition or kit provided, the reduced pharmacodynamically effective dose is at least 40% less. In any one other aspect of the method or composition or kit provided, the reduced pharmacodynamically effective dose is at least 50% less.

In any one aspect of the methods or compositions or kits provided, the immunosuppressive agent dose is a statin, mTOR inhibitor, TGF-β signaling agent, corticosteroid, mitochondrial function inhibitor, P38 inhibitor, NF-κβ. Includes inhibitors, adenosine receptor agonists, prostaglandin E2 agonists, phosphodiesterase 4 inhibitors, HDAC inhibitors or proteasome inhibitors. In any one other aspect of the provided method or composition or kit, the mTOR inhibitor is rapamycin.

In any one aspect of the methods or compositions or kits provided, the therapeutic macromolecule comprises a therapeutic protein. In any one other aspect of the provided method or composition or kit, the therapeutic macromolecule comprises a therapeutic polynucleotide. In any one other aspect of the provided method or composition or kit, the therapeutic protein is for protein replacement or protein augmentation therapy. In another aspect of any one of the methods or compositions or kits provided, the Therapeutic Polymers are instillable or injectable therapeutic proteins, enzymes, enzyme cofactors, hormones, blood or blood coagulation factors, Includes cytokines, interferons, growth factors, monoclonal antibodies, polyclonal antibodies, or proteins associated with Pompe's disease. In any one other aspect of the methods or compositions or kits provided, the therapeutic proteins that can be infused or injected are tocilizumab, alpha-1 antitrypsin, hematide, alfa interferon alfa-2b, Rhucin. , Tesamorelin, ocrelizumab, belimumab, pegloticase, pegsiticase, taliglucerase alpha, agarcidase alpha or velaglucerase alpha.

In another aspect of any one of the methods or compositions or kits provided, the enzyme comprises an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. In another aspect of any one of the methods or compositions or kits provided, the enzyme comprises an enzyme for enzyme replacement therapy for impaired lysosomal accumulation. In another aspect of any one of the methods or compositions or kits provided, the enzyme for enzyme replacement therapy for lysosomal accumulation disorders is imiglusulfase, a-galactosidase A (a-galA), agarsidase beta, acidic. Includes α-glucosidase (GAA), alglucosidase alfa, LUMIZYME, MYOZYME, arylsulfatase B, laronidase, ALDURAZYME, idursulfase, ELAPRASE, arylsulfase B or NAGLAZYME. In another aspect of any one of the methods or compositions or kits provided, the enzyme comprises KRYSTEXXA (pegloticase). In another aspect of any one of the methods or compositions or kits provided, the monoclonal antibody comprises HUMIRA (adalimumab).

In another aspect of any one of the methods or compositions or kits provided, cytokines include lymphokines, interleukins, chemokines, type 1 cytokines or type 2 cytokines. In any one other aspect of the provided method or composition or kit, the blood or blood coagulation factor is a factor I, a factor II, a tissue factor, a factor V, a factor VII, a factor VIII, a factor X. Factor IX, Factor X, Factor Xa, Factor XII, Factor XIII, von Villebrand factor, Precaricrane, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, Protein Z-related protease inhibitor (ZPI), plasminogen, alpha2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator Includes Inhibitor-2 (PAI2), Cancer Procoagrant or Epoetin Alpha. In any one other aspect of the provided method or composition or kit, the blood or blood coagulation factor is factor VIII.

In any one aspect of the methods or compositions or kits provided, the load of immunosuppressant attached to the synthetic nanocarrier is between 0.1% and 50% on average across the synthetic nanocarrier. Is. In any one other aspect of the provided method or composition or kit, the load capacity is between 0.1% and 20%.

In any one aspect of the methods or compositions or kits provided, synthetic nanoparticles are lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant emulsions, dendrimers, bucky balls, nanowires, virus-like. Includes particles or peptide or protein particles. In any one other aspect of the provided method or composition or kit, the synthetic nanocarrier comprises lipid nanoparticles. In any one other aspect of the provided method or composition or kit, the synthetic nanocarrier comprises liposomes. In any one other aspect of the provided method or composition or kit, the synthetic nanocarrier comprises metal nanoparticles. In any one other aspect of the provided method or composition or kit, the metal nanoparticles comprise gold nanoparticles. In any one other aspect of the provided method or composition or kit, the synthetic nanocarrier comprises polymer nanoparticles.

In any one other aspect of the provided method or composition or kit, the polymer nanoparticles comprise a polymer that is a non-methoxy-terminated pluronic polymer. In another aspect of any one of the methods or compositions or kits provided, the polymer nanoparticles are polyester, polyester attached to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethylene. Including imin. In another aspect of any one of the methods or compositions or kits provided, the polyester comprises poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid) or polycaprolactone. In another aspect of any one of the methods or compositions or kits provided, the polymer nanoparticles comprise a polyester and a polyester attached to a polyether. In another aspect of any one of the methods or compositions or kits provided, the polyether comprises polyethylene glycol or polypropylene glycol.

In any one aspect of the provided method or composition or kit, the average value of the particle size distribution obtained using dynamic light scattering of synthetic nanocarriers is greater than 100 nm in diameter. In any one other aspect of the provided method or composition or kit, the diameter is greater than 150 nm. In any one other aspect of the provided method or composition or kit, the diameter is greater than 200 nm. In any one other aspect of the provided method or composition or kit, the diameter is greater than 250 nm. In any one other aspect of the provided method or composition or kit, the diameter is greater than 300 nm.

In any one aspect of the methods or compositions or kits provided, the aspect ratios of the synthetic nanocarriers are 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1 Greater than: 5, 1: 7, or 1:10.

In another aspect, there is provided a method of making any one of the compositions or kits provided herein. In one aspect, the method of manufacture comprises producing a dose or dosage form of a Therapeutic macromolecule and producing a dose or dosage form of an immunosuppressive agent. In some embodiments, the therapeutic polymer dose or dosage form is a reduced pharmacodynamically effective dose of the therapeutic macromolecule. In another aspect of any one of the methods provided, the step of producing a dose or dosage form of an immunosuppressant comprises attaching the immunosuppressant to a synthetic nanocarrier. In another aspect of any one of the manufacturing methods provided, the method further comprises combining a dose or dosage form of an immunosuppressive agent with a dose or dosage form of a Therapeutic macromolecule in a kit.

In another aspect, the use of either of the compositions or kits provided herein is provided for the manufacture of a medicament for reducing an anti-therapeutic polymeric antibody response in a subject. In one aspect, the composition or kit comprises an immunosuppressant and a therapeutic macromolecule, wherein the therapeutic macromolecule may be provided in a reduced pharmacodynamically effective dose of the therapeutic macromolecule. In any one other aspect of the use provided herein, the immunosuppressant is attached to a synthetic nanocarrier. In another aspect of any one of the uses provided herein, the use is to achieve any one of the methods provided herein.

In another aspect, any one of the compositions or kits provided herein may be for use in any one of the methods provided herein. In one aspect, the composition or kit comprises one or more doses or dosage forms of therapeutic macromolecules and / or one or more doses or dosage forms of immunosuppressive agents. In one aspect, the therapeutic polymer dose is a reduced pharmacodynamically effective dose. In another embodiment, the immunosuppressant is attached to the synthetic nanoparticles.

In another aspect, methods of making pharmaceuticals intended to reduce anti-therapeutic polymeric antibody responses are provided. In one aspect, the medicament comprises an immunosuppressant and / or a therapeutic macromolecule, wherein the therapeutic macromolecule may be a reduced pharmacodynamically effective dose. In another aspect of any one of the production methods provided herein, the immunosuppressant is attached to a synthetic nanocarrier.

Brief description of the figure
FIG. 1 shows the level of antigen-specific circulating antibody production by concomitant administration provided herein. FIG. 2 shows the level of antigen-specific circulating antibody production by concomitant administration provided herein. FIG. 3 provides anti-OVA antibody titers with concomitant dosing provided herein. FIG. 4 provides anti-KLH antibody titers with concomitant administration provided herein. FIG. 5 shows the antibody recall response to the final nanocarrier and FVIII one month after the final dosing of FVIII.

6A and 6B show the effectiveness of nanocarrier and FVIII dosing in hemophilia A mice. 7A and 7B show the immune response to HUMIRA in mice treated with HUMIRA / adalimumab in the presence or absence of nanocarriers attached to rapamycin. FIG. 8 shows the anti-keyhole limpet hemocyanin (KLH) antibody titer in KLH-treated mice with and without nanocarriers attached to rapamycin. FIG. 9 shows anti-ovalbumin (OVA) antibody titers in OVA-treated mice with and without nanocarriers attached to rapamycin. FIG. 10 shows anti-KRYSTEXXA antibody titers in KRYSTEXXA-treated mice with and without nanocarriers attached to rapamycin.

FIG. 11 shows antibody titers in OVA and KLH treated mice, either in the presence or absence of nanocarriers attached to rapamycin. 12A and 12B show the immune response to KLH in KLH-treated mice with and without nanocarriers attached to rapamycin. 13A and 13B show the immune response to HUMIRA / adalimumab in mice treated with HUMIRA / adalimumab in the presence or absence of nanocarriers attached to rapamycin. FIG. 14 is a diagram that provides an exemplary protocol for performing the methods provided herein. FIG. 15 shows the beneficial effect of performing the methods provided herein with respect to treatment with HUMIRA.

FIG. 16 provides an exemplary protocol for performing the methods provided herein. FIG. 17 shows the beneficial effect of performing the methods provided herein with respect to treatment with HUMIRA. FIG. 18 demonstrates a reduction in anti-protein antibody response as a result of two different immunosuppressive agents attached to synthetic nanocarriers. FIG. 19 shows another exemplary protocol and beneficial effects of performing the methods provided herein for treatment with HUMIRA.

Detailed Description of the Invention Before discussing the invention in detail, it should be understood that the invention is not limited to such materials, as the specifically exemplified materials or process parameters can of course vary. .. It is also not intended that the terminology used herein is merely to describe specific aspects of the invention and is not intended to limit the use of alternative terminology to describe the invention. Should be understood.
All publications, patents and patent applications cited herein, either above or below, are incorporated herein by reference in their entirety for all purposes.

As used herein and in the accompanying claims, the singular "a," "an," and "the" include references unless otherwise expressly stated. For example, the reference to "a polymer" includes a mixture of two or more such molecules or a mixture of different molecular weights of a single polymer species, and the reference to "synthetic nanocarriers" is 2. Including a mixture of one or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, the reference to "RNA molecule" is a mixture of two or more such RNA molecules or multiple such RNA molecules. And references to "immunosuppressive agents" include two or more such materials or multiple such immunosuppressive agent molecules, etc.

As used herein, variants such as the term "comprise", or "comprises" or "comprising" are any listed integers (eg, features, elements, features, properties, etc.). It should be read as displaying the inclusion of a method / process step or limitation) or a group of perfections (eg, multiple features, elements, features, characteristics, methods / process steps or limitations), any other perfect or perfection. It should not be read as displaying the exclusion of body groups. Therefore, as used herein, the term "comprising" is comprehensive and does not exclude additional, unlisted completes or method / process steps.

In any one aspect of the compositions and methods provided herein, "comprising" is replaced by "consisting essentially of" or "consisting of". May be done. The phrase "consisting essentially" is used herein because it requires the specified completeness (s) or steps together with those that do not significantly affect the nature or function of the claimed invention. Will be done. As used herein, the term "consisting" refers to an enumerated complete (eg, feature, element, feature, characteristic, method / process step or limitation) or group of perfect (eg, multiple features). , Elements, features, characteristics, methods / process steps or limitations) are used to indicate the presence of only.

A. Preface The methods, compositions or kits provided herein are used to improve the pharmacodynamic effect (s) of therapeutic macromolecules in subjects in which an antibody response to the therapeutic macromolecule has begun. obtain. Accordingly, the methods or compositions or kits provided herein improve the pharmacodynamic effects (s) of therapeutic macromolecules that are otherwise reduced due to the anti-therapeutic macromolecular antibody response. Can be used to Without being bound by any particular theory, it is believed that the methods or compositions or kits provided may be used to reduce the undesired humoral immune response to therapeutic macromolecules. In some embodiments, the method, composition or kit is provided when the subject is tolerated to a Therapeutic macromolecule and the Therapeutic macromolecule is administered without the combined administration of the immunosuppressive drug dose provided. It can be used to reduce the unwanted immune response that may occur, and such doses may be administered in combination.

Such an undesired immune response can result in improved clearance of the therapeutic macromolecule, or other interference with the therapeutic activity of the therapeutic macromolecule. Therefore, the pharmacodynamic effects (s) of therapeutic macromolecules can be improved (s) as a result of the reduction of unwanted immune responses, and / or therapeutic macromolecules by the methods, compositions or kits provided. The same level of effect can be achieved using reduced doses of. Therefore, repeated doses of therapeutic macromolecules may be administered to the subject as another result of the reduction of the undesired immune response.

Unexpectedly and surprisingly, immunosuppressive agents adhere to synthetic nanocarriers, preferably in some embodiments, in combination with therapeutic macromolecules and in the presence of anti-therapeutic macromolecular antibody responses. It has been found that delivery, when done, can result in improved pharmacodynamic effects. For example, the combinations described above can help neutralize anti-therapeutic polymer-specific antibodies that interfere with the desired therapeutic effect of the therapeutic macromolecule. The methods, compositions or kits provided herein not only reduce the undesired immune response to therapeutic macromolecules in some embodiments, but also when administered therapeutic macromolecules alone (its). It also results in an increase in the desired therapeutic effect of the therapeutic macromolecule, which would otherwise be reduced) (as a result of an unwanted immune response to the therapeutic macromolecule).

Therefore, the methods, compositions or kits provided herein are generally increased to compensate for the undesired immune response to therapeutic macromolecules when administered without the effects of the inventions provided herein. It may allow the subject to obtain the therapeutic effect of the therapeutic macromolecule without having to increase the dose of the therapeutic macromolecule that would be. Surprisingly, the methods, compositions or kits provided herein allow a subject to be administered a reduced dose of therapeutic macromolecule and even achieve the same therapeutic effect.

Since the undesired immune response that occurs during a therapeutic procedure with a Therapeutic polymer can be countered by the methods, compositions or kits provided, the present invention causes an undesired immune response against a Therapeutic polymer. , Or to use reduced pharmacodynamically effective doses to achieve improved pharmacodynamic effects in subjects expected to occur. In any one aspect of the methods provided herein, a subject may be a subject at risk for such an unwanted immune response.
Here, the present invention will be described in more detail below.

B. Definitions By "administering" or "administering" or "administering" means providing material to a subject in a pharmacologically useful manner. The term is intended to include Causing to be administered in some embodiments. "To administer" means to directly or indirectly force, encourage, encourage, assist, induce, or instruct another party to administer the material. ..

An "effective amount" is the development of one or more desirable responses in a subject, such as a tolerant immune response (eg, specific to a Therapeutic Polymer), in the context of the composition or dose for administration to the subject. The amount or dose of composition that causes the growth, activation, induction, survival, reduction of mobilization, or reduction of production of therapeutic macromolecule-specific antibodies). In some embodiments, the effective amount is a pharmacodynamically effective amount. Therefore, in some embodiments, effective amounts are provided herein that produce one or more of the desired pharmacodynamic, therapeutic and / or immune responses provided herein. The amount or dose of any of the compositions (or the plurality of compositions or doses provided herein). This amount may be intended for in vitro or in vivo. For in vitro purposes, the amount is the amount that clinicians would consider to have clinical effects for patients requiring administration of therapeutic macromolecules and / or antigen-specific immunotolerance to them. obtain.

Effective amounts may be involved in reducing the level of unwanted immune responses, but in some embodiments it is involved in completely preventing unwanted immune responses. Effective amounts may also be involved in delaying the occurrence of unwanted immune responses. An effective amount can also be an amount that produces the desired therapeutic endpoint or desired therapeutic outcome. In other embodiments, the effective amount may be involved in improving the level of desired response, such as treatment endpoint or treatment outcome. Effective amounts preferably provide a tolerant immune response to antigens such as therapeutic macromolecules in the subject. Achievements of any of the above can be monitored by routine methods.

In some embodiments of any one of the methods provided, the effective amount is such that the desired response is at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months. An amount that lasts in the subject for at least 5 months or longer. In any other aspect of the compositions and methods provided, the effective amount provides a measurable and desirable response for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least. The amount produced for 4 months, at least 5 months, or longer.

The effective dose is, of course, within the knowledge and experience of the healthcare professional, the specific subject being treated; the severity of the condition, disease or disorder; individual patient parameters including age, physical condition, size and weight. Duration of treatment; characteristics of combination treatment (if present); will depend on specific route of administration and homologous factors. These factors are well known to those of skill in the art and can be dealt with by routine experimental methods. It is generally preferred to use the maximum dose, i.e. the highest safe dose according to sound medical judgment. However, it will be appreciated by those skilled in the art that patients can claim lower doses, or tolerable doses, for medical, psychological, or virtually any other reason.

Generally, the dose of immunosuppressant and / or therapeutic macromolecule in the composition of the invention refers to the amount of immunosuppressant and / or therapeutic macromolecule. Alternatively, the dose may be administered based on the number of synthetic nanocarriers that provide the desired amount of immunosuppressant and / or therapeutic macromolecule.

An "anti-therapeutic polymer antibody response" or "anti-therapeutic polymer-specific antibody response" is the development of anti-therapeutic polymer-specific antibodies or the production of such antibodies as a result of administration of a therapeutic polymer. It is the guidance of the process to do. In some embodiments, such response counteracts the therapeutic effect of therapeutic macromolecules.

"Antigen" means a B cell antigen or a T cell antigen. "Type of antigen (s)" means molecules that share the same or substantially the same antigenic characteristics. In some embodiments, the antigen can be a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, polysaccharide, or is contained or expressed in a cell. In some embodiments, such as when the antigen is not well defined or characterized, the antigen may be contained in a cell or tissue preparation, cell debris, cell exosomes, conditioned medium and the like.

"Antigen-specific" refers to any immune response that results from the presence of an antigen or a portion thereof, or that produces a molecule that specifically recognizes or binds to an antigen. In some embodiments, antigen-specific can mean therapeutic macromolecule-specific when the antigen comprises a Therapeutic macromolecule. For example, when the immune response is antigen-specific antibody production, such as therapeutic polymer-specific antibody production, an antibody that specifically binds to an antigen (eg, therapeutic polymer) is produced. As another example, if the immune response is the proliferation and / or activity of antigen-specific B cells or CD4 + T cells, the proliferation and / or activity is the antigen or a portion thereof, alone or in an MHC molecule, B. It is brought about by recognition in combination with cells and the like.

"Assessing a pharmacodynamic effect" refers to the measurement or determination of the level, presence or absence, reduction, increase, etc. of a pharmacodynamic effect in vitro or in vivo. Such measurements or determinations can be made on one or more samples obtained from the subject. Such an assessment may be provided herein or otherwise made in any one of the methods known in the art.

A "risk" subject has the opportunity to be considered by the healthcare professional to have the opportunity to have a disease, disorder or condition, or to experience the unwanted anti-therapeutic polymeric antibody response provided herein. , The subject of the medical practitioner's belief that the composition and method provided will be effective. In any one aspect of the methods, compositions or kits provided herein, a subject is a subject at risk of having an anti-therapeutic macromolecular antibody response to a therapeutic macromolecule. In another aspect of any one of the methods, compositions or kits provided herein, a subject is a subject expected to have an anti-therapeutic macromolecular antibody response to a therapeutic macromolecule.

"Attached" or "attached" or "connected" or "connected" (etc.) means that one entity (eg, a site) is chemically associated with another. In some embodiments, the attachment is covalent, which means that the attachment occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, non-covalent attachments are charge interactions, affinity interactions, metal coordination, physsisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bond interactions. It is mediated by non-covalent interactions, including, but not limited to, actions, van der Waals interactions, magnetic interactions, electrostatic interactions, bipolar-dipole interactions and / or combinations thereof. In some embodiments, encapsulation is a form of attachment.

In some embodiments, the therapeutic macromolecule and immunosuppressant do not adhere to each other, which is subject to a process specifically intended for the therapeutic macromolecule and immunosuppressant to chemically associate one with the other. It means that it will not be done. In some embodiments, the therapeutic macromolecule and / or immunosuppressant is not attached to the synthetic nanocarrier, which is that the therapeutic macromolecule (and / or immunosuppressant) and the synthetic nanocarrier chemically combine one with the other. It means that it is not subjected to a process specifically intended to be related.

"Average" as used herein means the arithmetic mean, unless otherwise noted.
When applied to two or more materials and / or agents (also referred to herein as constituents), a "combination" refers to the material to which the two or more materials / agents are associated. Intended to be defined. The components can be identified separately, for example, a first component, a second component, a third component, and the like. The terms "combined" and "combined" in this context shall be construed accordingly.

The association of two or more materials / agents in a combination may be physical or non-physical. Examples of physically related materials / agents include:
Compositions (eg, unit formulations) containing two or more materials / agents in an admixture (eg, within the same unit dose);
A composition comprising materials in which two or more materials / agents are chemically / physicochemically linked (eg, cross-linking, molecular aggregation or binding to a common vehicle site);
• Two or more materials / agents are chemically / physicochemically co-packaged (eg, on lipid vesicles, particles (eg micro or nanoparticles) or emulsified droplets or Composition containing the material (arranged within);
• A pharmaceutical kit, pharmaceutical pack or patient pack in which two or more materials / agents are co-packaged or co-presented (eg, as part of a series of unit doses).

Examples of non-physically related and combined materials / agents include:
An improvised association of at least one compound / agent to form a physical association of the two or more materials / agents with at least one of the two or more materials / agents. Ingredients (eg unit formulations), including with instructions for use;
A material (eg, a unit formulation) comprising at least one of two or more materials / agents together with instructions for combined treatment with the two or more materials / agents;
-Patient population to which at least one of two or more materials / agents has been administered (or has been administered) to the other (s) of the two or more materials / agents. Including with instructions for administration to;
• Specifically adapted to use at least one of the two or more materials / agents in combination with the other (s) of the two or more materials / agents. Material, including in quantity or form.

As used herein, the term "combination therapy" is intended to define a therapy that includes the use of two or more material / agent combinations (as defined above). Therefore, references to "combination therapy", "combination" and "combination" use of materials / agents in this application may refer to materials / agents administered as part of the same overall treatment regimen. Therefore, the quantification of each of the two or more materials / agents may be different and each may be administered simultaneously or at different times. Therefore, the combination materials / agents may be administered either sequentially (eg, before or after) or simultaneously, either in the same pharmaceutical formulation (ie together) or in different pharmaceutical formulations (ie separately). You will see that. Simultaneously in the same product means as a unified product, while in different pharmaceutical products, it means non-unified product at the same time. The dosage regimen of each of the two or more materials / agents in combination therapy may also differ with respect to the route of administration.

A "combination" is a time-sufficient correlation of two or more materials / agents to provide a time-correlated, preferably modulation in a physiological or immunological response. It means that the subject is administered in such a manner that the two or more materials / agents thereof are administered in combination. In some embodiments, the combination administration is two or three within the specified period, preferably within one month, more preferably within one week, even more preferably within one day, even more preferably within one hour. It can cover the administration of the above materials / drugs. In some embodiments, the material / agent may be administered in combination repeatedly, i.e., in combination at more than one occasion, such as the combination administration that may be provided in the example.

"Determining" or "determining" means confirming the facts. Determining can be performed in a number of ways, including but not limited to conducting experiments or making predictions. As an example, the dose of immunosuppressant or therapeutic macromolecule is determined by starting with the test dose and using known scaling techniques (such as arometric or isometric scaling) to determine the dose for administration. Can be done. Such may also be used to determine the protocol provided herein. In another embodiment, the dose may be determined by testing different doses in the subject, i.e. through direct experiments based on experience and guide data. In some embodiments, "determining" or "determining" includes "determining." "Determining" means inviting, encouraging, encouraging, assisting, guiding or instructing an entity to confirm facts, or acting in cooperation with that entity. Means, including directly or indirectly, or explicitly or implicitly.

"Dosage form" means a pharmacologically and / or immunologically active material in a vehicle, carrier, vehicle or device suitable for administration to a subject. Any one of the compositions or doses provided herein may be in the dosage form.
"Dose" refers to the specific quantity of a pharmacologically and / or immunologically active material to be administered to a subject over a given period of time.

"Encapsulating" means encapsulating at least a portion of a substance within a synthetic nanocarrier. In some embodiments, the material is completely encapsulated within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated material is not exposed to the external local environment of the synthetic nanocarrier. In other forms, exposure to the local environment does not exceed 50%, 40%, 30%, 20%, 10% or 5% (weight / weight). Encapsulation is distinguished from absorption (absorption places most or all of the material on the surface of the synthetic nanocarrier, leaving the material exposed to the local environment outside the synthetic nanocarrier).

"Generating" means causing an action such as a physiological or immunological response (eg, a tolerant immune response), either directly or indirectly, on its own.
"Identifying an object" is any action or set of actions that makes a clinician recognize an object as an object for which an effect can be obtained from the methods, compositions or kits provided herein. Preferably, the identified subject requires a therapeutic effect from the therapeutic macromolecules provided herein and has or is suspected of having an anti-therapeutic macromolecule-specific antibody response. Is. The action or set of actions may be direct or indirect in itself. In any one aspect of the methods provided herein, the method further comprises identifying an object in need of the methods, compositions or kits provided herein.

"Immunosuppressive agent" means a compound that causes APC to have an immunosuppressive effect or to suppress T cells or B cells (eg, a tolerant effect). Immunosuppressive effects generally refer to the production or expression of cytokines or other factors by APCs that reduce, inhibit or prevent unwanted immune responses, or promote desirable immune responses such as regulatory immune responses. When an APC acquires an immunosuppressive function (under an immunosuppressive effect) against immune cells that recognize the antigen presented by this APC, the immunosuppressive effect is said to be specific to the presented antigen. ..

Without being bound by any specific theory, the immunosuppressive effect appears to be the result of the immunosuppressive agent being delivered to the APC, preferably in the presence of the antigen. In one aspect, the immunosuppressant is one that promotes APC to a regulatory phenotype in one or more immune effector cells. For example, regulatory phenotypes include inhibition of antigen-specific CD4 + T cell or B cell production, induction, stimulation or recruitment, inhibition of antigen-specific antibody production, production, induction, stimulation or recruitment of Treg cells (eg, CD4 + CD25highFoxP3 + Treg cells). , Etc. can be characterized.

This may be the result of conversion of CD4 + T cells or B cells to a regulatory phenotype. This can also be the result of induction of FoxP3 in other immune cells such as CD8 + T cells, macrophages and iNKT cells. In one aspect, the immunosuppressant affects the response of the APC after processing the antigen. In another embodiment, the immunosuppressant does not interfere with the processing of the antigen. In a further embodiment, the immunosuppressant is not an apoptotic signal molecule. In another embodiment, the immunosuppressant is not a phospholipid.

Immunosuppressants include statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors such as tricostatin A; corticosteroids; mitokinetics such as rotenone. Functional inhibitors; P38 inhibitors; NF-κβ inhibitors such as 6Bio, dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists such as mysoprostol (PGE2); phosphodiesterase 4 inhibitors such as loliplum (PGE2) Phosphodiesterase inhibitors such as PDE4); proteasome inhibitors; kinase inhibitors; G protein-conjugated receptor agonists; G protein-conjugated receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors;

Cytokine receptor activator; peroxysome proliferator-activated receptor antagonist; peroxysome proliferator-activated receptor agonist; histone deacetylase inhibitor; calcinulin inhibitor; phosphatase inhibitor; PI3KB inhibitor such as TGX-221; 3-methyladenine and the like Oxidized ATP such as, but not limited to, autophagy inhibitors; arylhydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and P2X receptor blockers. Immunosuppressants also include cyclosporines such as IDO, vitamin D3, cyclosporin A, arylhydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-thioguanine). TG), FK506, sangryferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol, methotrexate, and tryptride. In some embodiments, the immunosuppressive agent may comprise any of the agents provided herein.

The immunosuppressive agent may be a compound that directly provides an immunosuppressive effect to APC, or a compound that indirectly provides an immunosuppressive effect (that is, after being treated in some way after administration). Thus, immunosuppressants include prodrug forms of any of the compounds provided herein.

In any one aspect of the methods, compositions or kits provided herein, the immunosuppressive agent provided herein is attached to a synthetic nanocarrier. In a preferred embodiment, the immunosuppressant is an additional element to the material constituting the structure of the synthetic nanocarrier. For example, in one embodiment in which the synthetic nanocarrier is composed of one or more polymers, the immunosuppressant is a compound that is added and attached to the one or more polymers. As another example, in one embodiment in which the synthetic nanocarrier is composed of one or more lipids, the immunosuppressant is also a compound added and attached to one or more lipids. .. In some embodiments, such as when the synthetic nanocarrier material also provides an immunosuppressive effect, the immunosuppressant is an element present in addition to the synthetic nanocarrier material that provides the immunosuppressive effect.

Other exemplary immunosuppressants include small molecule drugs, natural products, antibodies (eg, antibodies against CD20, CD3, CD4), biologics drugs, carbohydrate drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, etc. Examples include, but are not limited to, aptamar, methotrexate, NSAID; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506); cytokines and growth factors such as TGF-β and IL-10. Further immunosuppressive agents are known to those of skill in the art and the invention is not limited in this regard.

In any one aspect of the methods, compositions or kits provided herein, the immunosuppressant is in a form such as a nanocrystalline form, whereby the immunosuppressant itself is in the form of particles or particles. is there. In some embodiments, these forms mimic a virus or other foreign pathogen. Many drugs have been nanonized and suitable methods for producing these drug forms will be known to those of skill in the art. Drug nanocrystals such as nanocrystalline rapamycin are known to those of skill in the art (Katteboinaa, et al. 2009, International Journal of PharmTech Resesarch; Vol. 1, No. 3; pp682-694).

As used herein, "drug nanocrystal" refers to the form of a drug (eg, an immunosuppressant) that does not include a carrier or matrix material. In some embodiments, the drug-drug nanocrystal comprises 90%, 95%, 98% or 99%, or more of the drug. Methods for producing drug nanocrystals include, but are not limited to, milling, high pressure homogenization, precipitation, spray drying, supercritical solution rapid expansion (RESS), Nanoedge® technology (Baxter Healthcare) and Nanocrystal Technology. (Trademark) (Elan Corporation).

In some embodiments, surfactants or stabilizers may be used for steric or electrostatic stability of the drug nanocrystals. In some embodiments, the nanocrystalline or nanocrystalline form of the immunosuppressant is soluble, stable and / or bioutilized of the immunosuppressant, particularly an insoluble or labile immunosuppressant. Can be used to increase performance. In some embodiments, combined administration of a reduced pharmacodynamically effective dose of the Therapeutic Polymer with a nanocrystalline form of an immunosuppressant results in a reduced anti-therapeutic Polymer antibody response.

The "load" when attached to a synthetic nanocarrier is the weight of the immunosuppressant and / or therapeutic polymer attached to the synthetic nanocarrier based on the total dry formulation weight of the material throughout the synthetic nanocarrier. Amount (weight / weight). Generally, the load capacity is calculated as an average over a population of synthetic nanocarriers. In one aspect, the payload is between 0.1% and 99% on average across synthetic nanocarriers. In another aspect, the load capacity is between 0.1% and 50%. In another embodiment, the payload of the immunosuppressant and / or therapeutic macromolecule is between 0.1% and 20%. In a further embodiment, the payload of the immunosuppressant and / or therapeutic macromolecule is between 0.1% and 10%.

In yet a further embodiment, the payload of the immunosuppressant and / or therapeutic macromolecule is between 1% and 10%. In yet a further embodiment, the immunosuppressant loading is between 7% and 20%. In yet another embodiment, the load of immunosuppressive and / or therapeutic macromolecules averages at least 0.1%, at least 0.2%, at least 0.3%, at least across the population of synthetic nanocarriers. 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4% , At least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% , At least 96%, at least 97%, at least 98% or at least 99%.

In a further embodiment, the loadings of immunosuppressive and / or therapeutic macromolecules averaged 0.1%, 0.2%, 0.3%, 0.4% across the population of synthetic nanocarriers. 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In some of the above embodiments, the load of immunosuppressant and / or therapeutic macromolecules does not exceed 25% on average across the population of synthetic nanocarriers. In some embodiments, the load capacity is as described in the examples, or otherwise calculated as known in the art.

In some embodiments, when the form of the immunosuppressant is itself a particle or particle-like, such as a nanocrystalline immunosuppressant, the payload of the immunosuppressant is the amount (weight / weight / weight / weight / of) of the immunosuppressant in the particle or the like. Weight). In such embodiments, the load capacity can reach 97%, 98%, 99% or more.
"Maximum size of synthetic nanocarrier" means the longest size of the nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of synthetic nanocarrier" means the smallest dimension of synthetic nanocarrier measured along any axis of synthetic nanocarrier. For example, in a spheroidal synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier will be substantially the same and will be the size of their diameter. Similarly, in a cube-like synthetic nanocarrier, the minimum dimension of the synthetic nanocarrier is the smallest of its height, width or length, while the maximum dimension of the synthetic nanocarrier is its height, width or length. Will be the largest of them.

In one embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is 100 nm or greater. In one embodiment, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is 5 μm or less. Preferably, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably. Greater than 120 nm, more preferably greater than 130 nm, more preferably still greater than 150 nm.

The aspect ratio between the maximum and minimum dimensions of the synthetic nanocarrier can vary depending on the embodiment. For example, the aspect ratio of the maximum dimension of the synthetic nanocarrier to the minimum dimension is 1: 1 to 1,000,000: 1, preferably 1: 1 to 100,000: 1, more preferably 1: 1 to 10,000. It can vary from 1: 1, more preferably 1: 1 to 1000: 1, even more preferably 1: 1 to 100: 1, and even more preferably 1: 1 to 10: 1. Preferably, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 3 μm or less, more preferably 2 μm or less, based on the total number of synthetic nanocarriers in the sample. , More preferably 1 μm or less, more preferably 800 nm or less, more preferably 600 nm or less, still more preferably 500 nm or less.

In a preferred embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is 100 nm or more, more preferably 120 nm. As mentioned above, it is more preferably 130 nm or more, more preferably 140 nm or more, and even more preferably 150 nm or more. Measurement of synthetic nanocarrier dimensions (eg, effective dimensions) uses dynamic light scattering (DLS) (eg Brookhaven Zeta PALS instrument) in some embodiments where the synthetic nanocarrier is suspended in a liquid (usually aqueous) medium. May be obtained by using). For example, a suspension of synthetic nanocarriers can be diluted in pure water from an aqueous buffer to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01-0.1 mg / mL.

The diluted suspension may be prepared directly inside a suitable cuvette for DLS analysis or transferred to it. The cuvette can then be placed in DLS, equilibrated to a controlled temperature, and then scanned for a sufficient amount of time to obtain a stable and reproducible distribution based on appropriate inputs regarding the viscosity of the medium and the index of refraction of the sample. Then the average effective diameter or distribution is reported. Determining the effective size of a high aspect ratio or non-spheroidal synthetic nanocarrier may require augmentative techniques such as an electron microscope to obtain more accurate measurements. The "dimension" or "size" or "diameter" of a synthetic nanocarrier means the average value of the particle size distribution obtained, for example, using dynamic light scattering.

"Non-methoxy-terminated polymer" means a polymer having at least one end that terminates at a site other than methoxy. In some embodiments, the polymer has at least two ends that terminate at sites other than methoxy. In other embodiments, the polymer does not have a methoxy-terminated end. "Non-methoxy-terminated pluronic polymer" means a polymer other than a linear pluronic polymer having methoxy at both ends. The polymer nanoparticles provided herein can include non-methoxy-terminated polymers or non-methoxy-terminated pluronic polymers.

A "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is a pharmacologically inert material used with a pharmacologically active material to formulate a composition. Means material. Pharmaceutically acceptable excipients include sugars (eg glucose, lactose, etc.), preservatives such as antibacterial agents, reconstruction aids, colorants, saline (phosphate buffered saline, etc.) and buffers. Includes, but is not limited to, various materials known in the art.

The "pharmacodynamic effect" of a "pharmacodynamic response" means any physiological or immunological response as a result of administration of a Therapeutic macromolecule. Such a response can be a desirable response, such as one related to a therapeutic effect. The methods, compositions or kits provided herein, in some embodiments, have improved pharmacodynamics, such as improved therapeutic efficacy, when administered in the presence of an anti-therapeutic polymeric antibody response. It was found to have a therapeutic effect. In some examples, the improved pharmacodynamic effect is therapeutic when administered in the presence of an anti-therapeutic macromolecular antibody response, without the combined administration of the immunosuppressive agent doses provided herein. It is obtained with a therapeutic polymer dose equal to or less than the polymer dose.

Whether a material / agent is pharmacodynamically effective can be evaluated by standard methods. In some embodiments, the pharmacodynamic effects of using the methods, compositions or kits provided in the presence of an anti-therapeutic polymeric antibody response are also therapeutic in the presence of an anti-therapeutic polymeric antibody response. It is compared to the pharmacodynamic effect when the polymer is not so administered. In some embodiments, the comparison is made to the pharmacodynamic effect of the therapeutic macromolecule when administered alone in the presence of an anti-therapeutic macromolecular antibody response. In general, pharmacodynamic effects are assessed by administration in the presence of an anti-therapeutic polymeric antibody response, as it is desirable that the method, composition or kit be effective in overcoming such response. Therefore, the pharmacodynamic effect is determined when such a response is occurring.

"Protocol" means a pattern of administration to a subject and includes any dosing regimen of one or more substances to the subject. A protocol consists of elements (or variables); therefore, a protocol contains one or more elements. Such elements of the protocol may include dosage, dosing frequency, route of administration, dosing duration, dosing rate, dosing interval, any combination of the above, and the like. In some embodiments, such a protocol can be used to administer one or more compositions of the invention to one or more test subjects. The immune response in these test subjects can then be assessed to determine if the protocol is effective in producing the desired or desired level of pharmacodynamic effect.

Any other therapeutic and / or immune effect may also be assessed in lieu of or in addition to the immune response described above. One or more of the elements of the protocol may be pre-demonstrated in a test subject, such as a non-human subject, before being transferred to the human protocol. For example, doses demonstrated in non-human subjects may be scaled as an element of the human protocol using established techniques such as aromometric scaling or other scaling methods. Whether or not the protocol had the desired effect can be determined using either one of the methods provided herein or otherwise known in the art. For example, a sample is a specific composition provided herein to determine whether specific immune cells, cytokines, antibodies, etc. have been reduced, generated, activated, etc. It may be obtained from a subject administered according to a specific protocol.

Useful methods for detecting the presence and / or number of immune cells include, but are not limited to, flow cytometry (eg, FACS), ELISpot, proliferation response, cytokine production and immunohistochemistry. Antibodies and other binders for specific staining of immune cell markers are commercially available. Such kits typically include staining reagents for antigens that allow FACS-based detection, separation and / or quantification of the desired cell population from a heterogeneous population of cells. In some embodiments, a number of compositions provided herein are administered to another subject using one or more, or all or substantially all of the elements comprising the protocol. In some embodiments, the protocol has been demonstrated to provide reduced and / or improved pharmacodynamic effects on the antibody response to therapeutic macromolecules.

"Providing" means an action or set of actions performed by an individual that supplies the article or set or method of article required to carry out the invention. An action or set of actions can be taken directly or indirectly by itself.
"Providing a subject" means having a clinician contact the subject to administer the composition provided herein to the subject, or have the subject perform the methods provided herein. , Any action or set of actions. Preferably, the subject is a subject in need of administration of a Therapeutic macromolecule and antigen-specific immune tolerance to it. An action or set of actions can be taken directly or indirectly by itself. In any one aspect of the methods provided herein, the method further comprises providing an object.

"Recording improved pharmacodynamic effects" achieves improved pharmacodynamic effects at therapeutic polymer doses in the presence of real, expected, or suspected anti-therapeutic polymer antibody responses. It means making a note of what has been done, either in writing or in electronic form, or directly or indirectly inducing the expected activity for which such note will be made. Generally, in such situations, when administered without the immunosuppressive agent dose provided herein (eg, single dose) based on the information available at the time of the combination dose provided herein. It would not have been expected that the therapeutic polymer dose would achieve an improved pharmacodynamic effect in the presence of an anti-therapeutic polymer antibody response.

For example, in such situations, it is expected that the efficacy of therapeutic macromolecules would be reduced if administered without an immunosuppressive dose, but with the combination administration provided herein, Instead, an improvement in pharmacodynamic effects is observed. In some embodiments, recording occurs when an immunosuppressive drug dose combined with a therapeutic macromolecular dose is administered to the subject, or at any time thereafter. In some of these embodiments, the therapeutic polymer dose is reduced compared to the therapeutic polymer dose administered without an immunosuppressive drug dose in the presence of an anti-therapeutic polymer antibody response ( Or not exceed it). "Written form" as used herein refers to any record on a medium such as paper. "Electronic form" as used herein refers to any record on an electronic medium. Any one of the methods provided herein further comprises recording a therapeutic response and / or immune response in a subject to be treated according to the methods provided herein.

The "reduced pharmacodynamically effective dose" is compared to the amount of therapeutic macromolecule when not administered with the immunosuppressant dose (eg, when the therapeutic macromolecule is administered alone). Refers to a reduced amount of therapeutic macromolecule that can achieve similar pharmacodynamic effects when co-administered with the immunosuppressant dose provided herein. As used herein, a similar pharmacodynamic effect is the level of effect, which is within one log of another level measured in the same manner. Preferably, it does not exceed a 5-fold difference from similar pharmacodynamic effects. Even more preferably, similar pharmacodynamic effects do not exceed a two-fold difference.

"Subjects" are warm-blooded animals such as humans and primates; birds; pets or livestock such as cats, dogs, sheep, goats, cows, horses and pigs; experimental animals such as mice, rats and guinea pigs; fish; reptiles; Means animals including zoo and wild animals; etc.
"Synthetic nanocarrier (s)" means a discrete object that is not found in nature and possesses at least one dimension of 5 microns or less in size. Albumin nanoparticles are generally included as synthetic nanocarriers, but in certain embodiments, the synthetic nanoparticles do not include albumin nanoparticles. In some embodiments, the synthetic nanocarrier is chitosan-free. In other embodiments, the synthetic nanocarriers are not lipid nanoparticles. In a further embodiment, the synthetic nanocarrier is phospholipid-free.

Synthetic nanoparticles are one or more lipid-based nanoparticles (also referred to herein as lipid nanoparticles, ie nanoparticles in which most of the material constituting the structure is lipid), polymer nanoparticles, metal nanoparticles. , Surface active material emulsions, dendrimers, bucky balls, nanoparticles, virus-like particles (ie, particles composed primarily of viral structural proteins but not infectious or less infectious), peptides or protein-based particles (book) Also referred to herein as protein nanoparticles, i.e., using a combination of nanomaterials such as nanoparticles in which most of the materials that make up the structure are peptides or proteins (such as albumin nanoparticles) and / or lipid polymer nanoparticles. The nanoparticles may be developed, but are not limited to these.

Synthetic nanocarriers can have a variety of different shapes, including, but not limited to, spheroids, cubes, pyramids, ellipses, cylinders, donuts and the like. Synthetic nanocarriers according to the invention include one or more surfaces. Exemplary synthetic nanocarriers that may be adapted for use in the practice of the present invention are (1) biodegradable nanoparticles disclosed in US Pat. No. 5,543,158 (Gref et al.), (2) published. Polymer nanoparticles of US Patent Application No. 20060002852 (Saltzman et al.), (3) Nanoparticles constructed by lithography of US Patent Application No. 20090028910 (DeSimone et al.) Published, (4) International Application Publication No. Disclosure of 2009/051837 (von Andrian et al.), (5) Nanoparticles disclosed in published US Patent Application No. 2008/0145441 (Penades et al.),

(6) Protein nanoparticles disclosed in Published U.S. Patent Application No. 20090226525 (de los Rios et al.), (7) Published in U.S. Patent Application No. 20060222652 (Sebbel et al.) Virus-like particles, (8) Nucleic acid-attached virus-like particles disclosed in U.S. Patent Application No. 200605251677 (Bachmann et al.) Published, (9) Viruses disclosed in WO 2010047839A1 or 2009106999A2. Like Particles, (10) P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles", Nanomedicine. 5 (6): 843-853 (2010). Nano-precipitated nanoparticles,

(11) Apoptotic cells, apoptotic bodies, or synthetic or semi-synthetic mimics disclosed in US Patent Publication No. 2002/0086049, or (12) Look et al., "Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus" erythematosus in mice ", J. Clinical Investigation 123 (4): Includes those disclosed in 1741-1749 (2013). In some embodiments, the synthetic nanocarrier is greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10. Can own a large aspect ratio.

Synthetic nanocarriers according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not contain a surface with a hydroxyl group that activates complement, or are alternative hydroxyl groups that activate complement. Includes surfaces that are essentially from no part. In a preferred embodiment, a synthetic nanocarrier according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not contain a surface that substantially activates complement, or instead substantially contains complement. Includes surfaces that are essentially from non-activated sites. In a more preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not contain a surface that activates complement or, instead, from a site that does not activate complement. Including the surface that becomes essential. In some embodiments, the synthetic nanocarrier eliminates virus-like particles. In some embodiments, the synthetic nanocarrier is greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7, or greater than 1:10. Can have an aspect ratio.

"Therapeutic macromolecule" refers to any protein, carbohydrate, lipid or nucleic acid that may be administered to a subject and have a therapeutic effect. In some embodiments, administration of a Therapeutic Polymer to a subject can result in an undesired immune response involving the production of anti-therapeutic polymer-specific antibodies. As described herein, administration of a therapeutic macromolecule in combination with an immunosuppressant dose may improve the therapeutic efficacy of the therapeutic macromolecule, such as by reducing an undesired immune response to it. In some embodiments, the therapeutic macromolecule can be a therapeutic polynucleotide or a therapeutic protein.

"Therapeutic polynucleotide" means any polynucleotide or polynucleotide-based treatment that may be administered to a subject and have a therapeutic effect. Such treatments include gene silencing. Examples of such treatments are known in the art and include, but are not limited to, naked RNA, including, but not limited to, the forms of messenger RNA, modified messenger RNA and RNAi. Examples of other therapeutic polynucleotides are set forth herein. Therapeutic polynucleotides may be produced in cells, on cells or by cells, or may be obtained using cell-free methods or fully synthetic in vitro methods. Subjects therefore include any subject in need of treatment by any of the aforementioned. Such objects include objects that will receive any of the above.

"Therapeutic protein" means any protein or protein-based treatment that may be administered to a subject and have a therapeutic effect. Such treatments include protein replacement therapy and protein augmentation therapy. Such therapies also include administration of exogenous or exogenous proteins, antibody therapies and cell or cell-based therapies. Therapeutic proteins include, but are not limited to, enzymes, enzyme cofactors, hormones, blood coagulation factors, cytokines, growth factors, monoclonal antibodies, antibody-drug conjugates and polyclonal antibodies. Examples of other therapeutic proteins are described separately herein. Therapeutic proteins may be produced in cells, on cells, or by cells, obtained from such cells, or administered in the form of such cells. In some embodiments, the therapeutic protein is in mammalian cells, insect cells, yeast cells, bacterial cells, plant cells, transgenic animal cells, transgenic plant cells, etc., on these cells, or by these cells. Produced. Therapeutic proteins may be produced by genetic recombination in such cells. Therapeutic proteins may be produced in, on, or by the virus-transformed cells. Subjects therefore include any subject requiring treatment by any of the aforementioned. Such objects include objects that will receive any of the above.

A "APC presentable antigen of a Therapeutic polymer" produces a therapeutic polymer (ie, an immune response to a Therapeutic polymer or a Therapeutic polymer (eg, production of an anti-therapeutic polymer-specific antibody). It means an antigen associated with the obtained fragment). In general, therapeutic polymeric antigen presenting cell (APC) presentable antigens include those presented by the immune system, including, but not limited to, dendritic cells, B cells or macrophages. Can be presented for recognition by cells of the immune system). APC-presentable antigens of therapeutic macromolecules can be presented, for example, for recognition by T cells. Such antigens can be recognized by T cells and trigger an immune response in T cells through the presentation of an epitope of the antigen bound to a Class I or Class II major histocompatibility complex molecule (MHC).

APC presentable antigens of therapeutic macromolecules generally include proteins, polypeptides, peptides, polynucleotides, lipoproteins, or are contained or expressed in, on, or by cells. Therapeutic polymeric antigens are attached to synthetic nanocarriers in some embodiments and include MHC class I-restricted epitopes and / or MHC class II-restricted epitopes and / or B cell epitopes. Preferably, one or more tolerant immune responses specific for therapeutic macromolecules are provided by the methods, compositions or kits provided herein. In some embodiments, the population of synthetic nanocarriers does not contain the APC-presentable antigen of the added therapeutic polymer, which means that during the production of the synthetic nanocarrier, a substantial amount of the therapeutic polymer. This means that the APC presentable antigen is not intentionally added to the synthetic nanocarrier.

An "undesirable immune response" is brought about by exposure to an antigen and promotes or exacerbates the diseases, disorders or conditions (or signs thereof) provided herein, or is provided herein. An undesired immune response that is a sign of a disease, disorder or condition. Such an immune response generally has an adverse effect on the health of the subject or is a sign of an adverse effect on the health of the subject. Undesirable immune responses include the production of antigen-specific antibodies, the proliferation and / or activity of antigen-specific B cells, or the proliferation and / or activity of antigen-specific CD4 + T cells. In general, these undesired immune responses are specific to therapeutic macromolecules and can counteract the desirable beneficial effects of administration of therapeutic macromolecules. Thus, in some embodiments, the undesired immune response is an anti-therapeutic polymeric antibody response.

C. Compositions and Related Methods Compositions herein include compositions containing immunosuppressants and therapeutic macromolecules and related methods or kits. Such methods, compositions or kits are treated in the presence of an anti-therapeutic macromolecular antibody response, such as by reducing or inhibiting an undesired immune response specific to the therapeutic macromolecule, which reduces the therapeutic effect of the therapeutic macromolecule. It is useful for improving the pharmacodynamic effect of macromolecules. Such methods, compositions or kits are also useful to allow repeated administration of therapeutic macromolecules. Therefore, the methods, compositions or kits provided herein are useful for achieving or enhancing the desired therapeutic effect of therapeutic macromolecules. In some embodiments, such therapeutic effects can be achieved or enhanced with reduced pharmacodynamically effective doses. The methods, compositions or kits provided may be useful for any subject who requires the therapeutic effect of a therapeutic macromolecule.

As mentioned above, delivering immunosuppressive agents in combination with therapeutic macromolecules in the presence of anti-therapeutic macromolecular antibody responses is preferably improved when attached to synthetic nanocarriers in some embodiments. It has been found that it can provide the desired pharmacodynamic effects, including the enhancement of such effects that can be achieved even at reduced doses of therapeutic macromolecules. For example, the method, composition or kit helps neutralize an anti-therapeutic polymer-specific antibody that interferes with the desired therapeutic effect of the therapeutic macromolecule. The methods, compositions or kits provided herein are therefore reduced when the Therapeutic Polymer is administered without an immunosuppressive dose (eg, when the Therapeutic Polymer is administered alone). It can result in a desired improvement in the therapeutic effect of the therapeutic macromolecules that will be produced.

Therefore, in some embodiments, the methods, compositions or kits provided herein allow a subject to obtain a therapeutic effect on a Therapeutic Polymer without the need to increase the dose of the Therapeutic Polymer. The dose will generally be increased to compensate for the undesired immune response to therapeutic macromolecules when administered without the effects of the inventions provided herein. Surprisingly, the methods, compositions or kits provided herein are the same, subject to administration of reduced doses of therapeutic macromolecules in the presence of an anti-therapeutic macromolecular antibody response. Or even make it possible to achieve better therapeutic effects.

In carrying out the present invention, various immunosuppressive agents may be used, which are preferably attached to synthetic nanocarriers. A wide variety of synthetic nanocarriers can be used according to the present invention. In some embodiments, the synthetic nanocarrier is spherical or spheroidal. In some embodiments, the synthetic nanocarrier is flat or flat. In some embodiments, the synthetic nanocarrier is cubic or cubic. In some embodiments, the synthetic nanocarrier is oval or oval. In some embodiments, the synthetic nanocarrier is a cylinder, cone or pyramid.

In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform in size or shape so that each synthetic nanocarrier has similar properties. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90% or at least 95% of synthetic nanocarriers have a minimum size that falls within 5%, 10% or 20% of the average diameter or average size of the synthetic nanocarriers. Or it may have the maximum dimensions.

The synthetic nanocarrier may be solid or hollow and may include one or more layers. In some embodiments, each layer has its own composition and unique properties relative to the other layers (s). For example, synthetic nanocarriers have a core / shell structure in which the core is one layer (eg, a polymer core) and the shell is a second layer (eg, a lipid bilayer or a lipid monolayer). You may. The synthetic nanocarrier may contain a plurality of different layers.

In some embodiments, the synthetic nanocarrier optionally comprises one or more lipids. In some embodiments, the synthetic nanocarrier may comprise liposomes. In some embodiments, the synthetic nanocarrier may comprise a lipid bilayer. In some embodiments, the synthetic nanocarrier may comprise a lipid monomolecular layer. In some embodiments, the synthetic nanocarrier may comprise micelles. In some embodiments, the synthetic nanocarrier may include a core comprising a polymer matrix surrounded by a lipid layer (eg, lipid bilayer, lipid monolayer, etc.). In some embodiments, the synthetic nanocarrier is a non-polymeric core (eg, metal particles, quantum dots, ceramic particles, bone particles, etc.) surrounded by a lipid layer (eg, lipid bilayer, lipid monolayer, etc.). It may contain virus particles, proteins, nucleic acids, carbohydrates, etc.).

In other embodiments, the synthetic nanocarrier may include metal particles, quantum dots, ceramic particles, and the like. In some embodiments, the non-polymer synthetic nanocarrier is an aggregate of non-polymeric constituents, such as an aggregate of metal atoms (eg, gold atoms).

In some embodiments, the synthetic nanocarrier may optionally include one or more amphipathic entities. In some embodiments, the amphipathic entity may facilitate the production of synthetic nanocarriers with increased stability, improved homogeneity, or increased viscosity. In some embodiments, the amphipathic entity may be associated with the inner surface of a lipid membrane (eg, lipid bilayer, lipid monolayer, etc.). Many amphipathic entities known in the art are suitable for use in making synthetic nanocarriers according to the present invention. Such amphipathic entities include phosphoglycerides;

Phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; aliphatic alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surface active fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; Polysorbate trioleate (Span® 85) glycocholate; sorbitan monolaurate (Span® 20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate 65 (Tween® 65); Polysorbate 80 (Tween® 80); Polysorbate 85 (Tween® 85); Polyoxyethylene monostearate; Surfactin; poloxomer; Polysorbate trioleate Such as sorbitan fatty acid ester; lecithin;

Risolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (kephalin); cardiolipin; phosphatidylic acid; celebroside; disetyl phosphate; dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; acetylpalmitate; Nolate; Hexadecyl stearate; Isopropyl millistate; Tyroxapol; Poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; Phospholipid; Synthesis with high surfactant activity and / or Natural detergents; deoxycholates; cyclodextrins; chaotropic salts; ion pairing agents; and combinations thereof, but are not limited thereto. The amphipathic entity component may be a mixture of different amphipathic entities. Those skilled in the art will recognize that this is a non-comprehensive and exemplary list of substances with surfactant activity. Any amphipathic entity may be used in the production of synthetic nanocarriers used in accordance with the present invention.

In some embodiments, the synthetic nanocarrier may optionally contain one or more carbohydrates. Carbohydrates may be natural or synthetic. The carbohydrate may be an induced natural carbohydrate. In certain embodiments, carbohydrates include glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cerbiose, mannose, xylose, arabinose, gluconic acid, galactonic acid, mannuronic acid, glucosamine, galatosamine and neuroamic acid. However, including, but not limited to, monosaccharides or disaccharides.

In certain embodiments, the carbohydrates are purulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose. , Chitosan, Ν, Ο-carboxymethyl chitosan, argin and alginic acid, starch, chitin, inulin, konjac, glucomannan, cellulose, heparin, hyaluronic acid, curdran and xanthan, but not limited to these polysaccharides. In some embodiments, the synthetic nanocarrier is free of (or specifically eliminated) carbohydrates such as polysaccharides. In certain embodiments, the carbohydrates may include carbohydrate derivatives such as sugar alcohols, including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol and lactitol.

In some embodiments, the synthetic nanocarrier may comprise one or more polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy-terminated pluronic polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymers constituting the synthetic nanocarrier. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (weight / weight) are non-methoxy-terminated pluronic It is a polymer.

In some embodiments, all of the polymers that make up the synthetic nanocarrier are non-methoxy-terminated pluronic polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy-terminated polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymers constituting the synthetic nanocarrier. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (weight / weight) non-methoxy-terminated polymers Is. In some embodiments, all of the polymers that make up the synthetic nanocarrier are non-methoxy-terminated polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that are free of pluronic polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymers constituting the synthetic nanocarrier. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (weight / weight) do not contain pluronic polymers. .. In some embodiments, all of the polymers that make up the synthetic nanocarriers are free of pluronic polymers. In some embodiments, the polymer may be surrounded by a coating layer (eg, liposomes, lipid monolayers, micelles, etc.). In some embodiments, the various elements of the synthetic nanocarrier may be attached to the polymer.

Immunosuppressants and / or therapeutic macromolecules can be attached to synthetic nanocarriers by any of many methods. In general, attachment can be the result of binding between immunosuppressants and / or therapeutic macromolecules and synthetic nanocarriers. This binding can result in immunosuppressants and / or therapeutic macromolecules being attached (encapsulated) to the surface of the synthetic nanocarrier and / or contained within the synthetic nanocarrier. However, in some embodiments, the immunosuppressant and / or therapeutic macromolecule is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than binding to the synthetic nanocarrier. In a preferred embodiment, the synthetic nanocarrier comprises the polymer provided herein and the immunosuppressant and / or therapeutic polymer is attached to the polymer.

When attachment occurs as a result of binding between immunosuppressants and / or therapeutic macromolecules and synthetic nanocarriers, attachment can occur via the link site. The linking site can be any site through which the immunosuppressant and / or therapeutic macromolecule is bound to the synthetic nanocarrier. Such sites include covalent bonds such as amide or ester bonds and separate molecules that (covalently or non-covalently) bind immunosuppressants and / or therapeutic macromolecules to synthetic nanocarriers. To. Such molecules include linkers, or polymers or units thereof. For example, the linking site may include a charged polymer to which an immunosuppressant and / or therapeutic polymer is electrostatically bound. As another example, the linking site may include a polymer or a unit thereof to which it is covalently attached.

In a preferred embodiment, the synthetic nanocarrier comprises the polymer provided herein. These synthetic nanocarriers may be completely polymeric or may be a mixture of the polymer and other materials.

In some embodiments, the polymers of the synthetic nanocarriers are associated with each other to form a polymer matrix. In some of these embodiments, components such as immunosuppressants or therapeutic macromolecules may be covalently associated with one or more polymers in the polymer matrix. In some embodiments, the covalent association is mediated by the linker. In some embodiments, the constituents may be non-covalently associated with one or more polymers in the polymer matrix. For example, in some embodiments, the constituents can be encapsulated within a polymeric matrix, surrounded by it, and / or dispersed throughout it. Alternatively or additionally, the constituents may be associated with one or more polymers in the polymer matrix by hydrophobic interactions, charge interactions, van der Waals forces, and the like. A wide variety of polymers and methods for forming polymer matrices are conventionally known.

The polymer can be a natural or non-natural (synthetic) polymer. The polymer can be a homopolymer or a copolymer containing two or more monomers. In terms of alignment, the copolymer may be random, block, or may include a combination of random and block alignment. Typically, the polymer according to the invention is an organic polymer.

In some embodiments, the polymer comprises polyester, polycarbonate, polyamide or polyether, or units thereof. In other embodiments, the polymer comprises poly (ethylene glycol) (PEG), polypropylene glycol, poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid) or polycaprolactone, or units thereof. Including. In some embodiments, the polymer is preferably biodegradable. Therefore, in these embodiments, if the polymer comprises a polyether such as poly (ethylene glycol) or polypropylene glycol or a unit thereof, the polymer is a block copolymer of the polyether and biodegradable such that it is biodegradable. It preferably contains a polymer. In other embodiments, the polymer does not include, by itself, polyethers or units thereof, such as poly (ethylene glycol) or polypropylene glycol or units thereof.

Other examples of polymers suitable for use in the present invention include polyesters, polycarbonates (eg poly (1,3-dioxane-2 on)), polyacid anhydrides (eg poly (sevacinic acid anhydride)), and the like. Polypropyl fumarate, polyamide (eg polycaprolactam), polyacetal, polyether, polyester (eg polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxyic acid (eg poly (β-hydroxyalkanoate))), Poly (orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polyurea, polystyrene and polyamine, polylysine, polylysine-PEG copolymer and poly (ethyleneimine), poly Examples include, but are not limited to, (ethyleneimine) -PEG copolymers.

In some embodiments, the polymer according to the invention is a polyester (eg, polylactic acid, poly (lactic acid-co-glycolic acid), polycaprolactone, polyvalerolactone, poly (1,3-dioxane-2one)); poly. The United States includes, but is not limited to, acid anhydrides (eg, poly (sebacic acid anhydride)); polyesters (eg, polyethylene glycol); polyurethanes; polymethacrylates; polyacryllates; and polycyanoacryllates. Includes polymers certified for use in humans under 21 C.FR §177.2600 by the Pharmaceutical Authority (FDA).

In some embodiments, the polymer can be hydrophilic. For example, the polymer may contain anionic groups (eg, phosphate groups, sulfate groups, carboxylate groups); cationic groups (eg, quaternary amine groups); or polar groups (eg, hydroxyl groups, thiol groups, amine groups). In some embodiments, the synthetic nanocarrier containing the hydrophilic polymer matrix creates a hydrophilic environment within the synthetic nanocarrier. In some embodiments, the polymer can be hydrophobic. In some embodiments, the synthetic nanocarrier containing the hydrophobic polymer matrix creates a hydrophobic environment within the synthetic nanocarrier. The choice of hydrophilicity or hydrophobicity of the polymer can affect the properties of the material incorporated (eg, attached) within the synthetic nanocarrier.

In some embodiments, the polymer can be modified with one or more sites and / or functional groups. Various sites or functional groups can be used according to the present invention. In some embodiments, the polymer can be modified with polyethylene glycol (PEG), with carbohydrates and / or with acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786: 301). Certain embodiments may be made using the general teachings of US Pat. No. 5,543,158 (Gref et al.) Or WO 2009/051837 (Von Andrian et al.).

In some embodiments, the polymer can be modified with lipid or fatty acid groups. In some embodiments, the fatty acid group is one or more of butyric acid, caproic acid, caprylic acid, caproic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid or lignoceric acid. obtain. In some embodiments, the fatty acid group is palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, gadrainic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid or erucic acid. Can be one or more of.

In some embodiments, the polymer comprises a lactic acid and glycolic acid unit, such as poly (lactic acid-co-glycolic acid) and poly (lactide-co-glycolide), collectively referred to herein as "PLGA". Copolymers; and homopolymers containing glycolic acid units referred to herein as "PGA"; and poly-L-lactic acid, poly-D-lactic acid, poly-D, L collectively referred to herein as "PLA". It may be a polyester containing-lactic acid, poly-L-lactide, poly-D-lactide and a homopolymer containing lactic acid units such as poly-D, L-lactide. In some embodiments, exemplary polyesters include, for example, polyhydroxyic acids; PEG copolymers and copolymers of lactide and glycolide (eg, PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers and derivatives thereof). Can be mentioned. In some embodiments, the polyester is, for example, poly (caprolactone), poly (caprolactone) -PEG copolymer, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4-hydroxy-L). -Proline ester), poly [α- (4-aminobutyl) -L-glycolic acid] and derivatives thereof.

In some embodiments, the polymer is PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and the lactic acid: glycolic acid ratio characterizes various forms of PLGA. Lactic acid can be L-lactic acid, D-lactic acid or D, L-lactic acid. The rate of decomposition of PLGA can be regulated by varying the lactic acid: glycolic acid ratio. In some embodiments, the PLGA used in accordance with the present invention is approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15: It is characterized by an 85 lactic acid: glycolic acid ratio.

In some embodiments, the polymer may be one or more acrylic polymers. In certain embodiments, the acrylic polymer is, for example, a copolymer of acrylic acid and methacrylic acid, a methylmethacrylate copolymer, an ethoxyethylmethacrylate, a cyanoethylmethacrylate, an aminoalkylmethacrylate copolymer, a poly (acrylic acid), a poly (methacrylic acid). , Methacrylic acid alkylamide copolymer, poly (methylmethacrylate), poly (anhydrous methacrylate), methylmethacrylate, polymethacrylate, poly (methylmethacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidylmethacrylate copolymer , Polycyanoacrylicates and combinations comprising one or more of the above polymers. Acrylic polymers can include fully polymerized copolymers of acrylic and methacrylic acid esters with low quaternary ammonium group content.

In some embodiments, the polymer can be a cationic polymer. Cationic polymers are generally capable of concentrating and / or protecting negatively charged strands of nucleic acid. Poly (lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; Kabanov et al., 1995, Bioconjugate Chem., 6: 7), poly (ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92: 7297) and poly (amide amine) dendrimer (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93) Amine-containing polymers such as: 4897; Tang et al., 1996, Bioconjugate Chem., 7: 703; Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively charged at physiological pH. Form an ion pair with the nucleic acid. In some embodiments, the synthetic nanocarrier may be free (or excluded) of the cationic polymer.

In some embodiments, the polymer is a biodegradable polyester with a cationic side chain (Putnam et al., 1999, Macromolecules, 32: 3658; Barrera et al., 1993, J, Am. Chem. Soc., 115. : 11010; Kwon et al., 1989, Macromolecules, 22: 3250; Lim et al., 1999, J. Am. Chem. Soc., 121: 5633; Zhou et al., 1990, Macromolecules, 23: 3399) possible. Examples of these polyesters are poly (L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010), poly (serine ester) (Zhou et. al., 1990, Macromolecules, 23: 3399), Poly (4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32: 3658; Lim et al., 1999, J. Am. Chem. Soc., 121: 5633) and poly (4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32: 3658; Lim et al., 1999, J. Am. Chem. Soc., 121 : 5633).

The properties of these and other polymers and their preparation methods are well known in the art (eg, US Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123: 9480; Lim et al., 2001, J. Am. Chem. Soc., 123: 2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62: 7; Uhrich et al ., 1999, Chem. Rev., 99: 3181). More generally, various methods for synthesizing certain suitable polymers include Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. By Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390: 386; and US Pat. Nos. 6,506,577, 6,632,922, 6,686,446. It is described in No. 6818,732 and No. 6,818,732.

In some embodiments, the polymer can be a linear or branched polymer. In some embodiments, the polymer can be a dendrimer. In some embodiments, the polymers may be substantially crosslinked with each other. In some embodiments, the polymer may be substantially free of crosslinks. In some embodiments, the polymer can be used according to the present invention without undergoing a cross-linking step. It should be further understood that synthetic nanocarriers may include block copolymers, graft copolymers, blends, mixtures and / or adducts of any of the aforementioned and other polymers. Those skilled in the art will recognize that the polymers listed herein represent a list of non-comprehensive exemplary polymers that may be used in accordance with the present invention.

In some embodiments, the synthetic nanocarrier is free of polymer constituents. In some embodiments, the synthetic nanocarrier may include metal particles, quantum dots, ceramic particles and the like. In some embodiments, the non-polymer synthetic nanocarrier is an aggregate of non-polymeric constituents, such as an aggregate of metal atoms (eg, gold atoms).

Compositions according to the invention may include the elements in combination with pharmaceutically acceptable excipients such as preservatives, buffers, saline or phosphate buffered saline. The composition can be made using conventional pharmaceutical manufacturing and compounding techniques to reach a useful dosage form. In some embodiments, compositions, such as those containing synthetic nanocarriers, are suspended in sterile aqueous saline for injection with preservatives.

In some embodiments, when preparing a synthetic nanocarrier as a carrier, a method of attaching components to the synthetic nanocarrier may be useful. If the component is a small molecule, it may be advantageous to attach the component to the polymer prior to assembly of the synthetic nanocarrier. In some embodiments, the components are attached to the polymer and then the polymer composite is used to attach the components to the synthetic nanocarrier through the use of their surface groups, rather than being used to construct synthetic nanocarriers. It may also be advantageous to prepare synthetic nanocarriers with surface groups to be treated.

In certain embodiments, the attachment can be made by a covalent linker. In some embodiments, the constituents according to the invention are by a 1,3-dipole cycloaddition of an azide group on the surface of the nanocarrier and a constituent containing an alkyne group, or by an alkyne and azido group on the surface of the nanocarrier. It can be covalently attached to the outer surface via the 1,2,3-triazole linker formed by the 1,3-dipole cycloaddition reaction with the constituents containing. Such a cycloaddition reaction is preferably carried out in the presence of a Cu (I) catalyst with a suitable Cu (I) ligand and a reducing agent for reducing the Cu (II) compound to a catalytically active Cu (I) compound. It is said. This Cu (I) catalytic azide-alkyne cycloaddition (CuAAC) is also sometimes referred to as a click reaction.

In addition, covalent attachment includes covalent linkers including amide linkers, disulfide linkers, thioether linkers, hydrazone linkers, hydrazidlinkers, imine or oxime linkers, urea or thiourea linkers, amidine linkers, amine linkers and sulfonamide linkers. May include.
The amide linker is formed via an amide bond between the amine on one component and the carboxylic acid group of the second component, such as a nanocarrier. The amide bond in the linker can be made using any of the conventional amide bond forming reactions with a preferably protected amino acid and an activated carboxylic acid (such as N-hydroxysuccinimide activated ester).

Disulfide linkers can be made, for example, through the formation of disulfide (SS) bonds between two sulfur atoms in the form of R1-SS-R2. A disulfide bond comprises a component containing a thiol / mercaptan group (-SH) and another activated thiol group on a polymer or nanocarrier, or a nanocarrier containing a thiol / mercaptan group and an activated thiol group. It can be formed by thiol exchange with the constituents it contains.

（式中Ｒ_１およびＲ_２が、いずれかの化学的実体であり得る）の１，２，３−トリアゾールは、ナノ担体などの第１構成成分に付着されたアジドの、免疫抑制剤または治療用高分子などの第２構成成分に付着された末端アルキンとの１，３−双極子環状付加反応により作られる。１，３−双極子環状付加反応は、触媒の有無で、好ましくはＣｕ（Ｉ）触媒を使用して、行われ、これにより２つの構成成分が１，２，３−トリアゾール官能性を通してつながれる。この化学は、Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) および Meldal, et al, Chem. Rev., 2008, 108(8), 2952-3015により詳細に記載され、「クリック」反応またはＣｕＡＡＣと呼ばれることも多い。 Triazole linker, specifically, the following forms

1,2,3-triazole (where R ₁ and R _{2 in the} formula can be any chemical entity) is an immunosuppressant or therapeutic agent for azides attached to a first component such as a nanocarrier. It is produced by a 1,3-dipole cycloaddition reaction with a terminal alkyne attached to a second component such as a polymer for use. The 1,3-dipole cycloaddition reaction is carried out with or without a catalyst, preferably using a Cu (I) catalyst, which connects the two components through 1,2,3-triazole functionality. .. This chemistry is detailed in Sharpless et al., Angew. Chem. Int. Ed. 41 (14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108 (8), 2952-3015. Often referred to as a "click" reaction or CuAAC.

In some embodiments, a polymer containing an azide or alkyne group at the end of the polymer chain is prepared. The polymer is then used to prepare synthetic nanocarriers in such a manner that multiple alkyne or azide groups are located on the surface of the nanocarrier. Alternatively, the synthetic nanocarrier may be prepared by another route and subsequently functionalized with an alkyne or azide group. The constituents are prepared by the presence of either an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne) group. The constituents are then reacted with the nanocarrier via a 1,3-dipole cycloaddition reaction in the presence or absence of a catalyst, which allows the constituents to become particles through the 1,4-disubstituted 1,2,3-triazole linker. Adhered in a covalent bond.

The thioether linker is made, for example, by the formation of a sulfur-carbon (thioether) bond in the form of R1-S-R2. Thioethers are made by alkylating a thiol / mercaptan (-SH) group on one component with an alkylating group such as a halide or epoxide on the second component. The thioether linker can also be formed by Michael addition of a thiol / mercaptan group on one component to an electron-deficient alkene group on a second component containing a maleimide or vinylsulfone group as a Michael acceptor. .. In another scheme, the thioether linker can be prepared by a radical thiol-ene reaction of a thiol / mercaptan group on one component with an alkene group on the second component.

The hydrazone linker is made by the reaction of a hydrazide group on one component with an aldehyde / ketone group on the second component.
The hydrazidlinker is formed by the reaction of a hydrazine group on one component with a carboxylic acid group on the second component. Such reactions are generally carried out using chemistry similar to the formation of amide bonds when the carboxylic acid is activated with an activating reagent.
The imine or oxime linker is formed by the reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on the second component.

Urea or thiourea linkers are prepared by the reaction of an amine group on one component with an isocyanate or thioisocyanate group on the second component.
The amidine linker is prepared by reacting an amine group on one component with an imide ester group on the second component.
Amine linkers are made by the alkylation reaction of an amine group on one component with an alkylating group such as a halide, epoxide or sulfonate ester group on the second component. Alternatively, the amine linker is also a suitable reducing reagent, such as sodium cyanoborohydride or sodium triacetoxyborohydride, with an amine group on one component and an aldehyde or ketone group on the second component. May be made by reductive amination using.

Sulfonamide linkers may be made by reacting an amine group on one component with a sulfonyl halide (such as a sulfonyl chloride) group on the second component.
Sulfone linkers are made by Michael addition of nucleophilic groups to vinyl sulfones. Either the vinyl sulfone or the nucleophilic group may be on the surface of the nanocarrier or may be attached to the constituents.
The components may also be complexed to the nanocarrier via a non-covalent complexing method. For example, a negatively charged therapeutic macromolecule or immunosuppressant may be conjugated to a positively charged nanocarrier through electrostatic adsorption. The components containing the metal ligand may also be complexed to the nanocarrier containing the metal complex via the metal-ligand complex.

In some embodiments, the constituents may be attached to a polymer such as polylactic acid-block-polyethylene glycol prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier may be reactive or activated on its surface. It may be formed so as to have a specific group. In the latter case, the constituents may be prepared to have groups compatible with the adhesion chemistry presented by the surface of the synthetic nanocarrier. In other embodiments, the peptide component may be attached to the VLP or liposome using a suitable linker. A linker is a compound or reagent that can attach two molecules to each other. In one aspect, the linker may be a homobifunctional or heterobifunctional reagent described in Hermanson 2008.

For example, a VLP or liposome synthetic nanocarrier containing a carboxyl group on its surface is treated with a homobifunctional linker, adipic acid dihydrazide (ADH) in the presence of EDC to form a corresponding synthetic nanocarrier with an ADH linker. Can be done. The resulting ADH-linked synthetic nanocarrier is then complexed with an acid group-containing peptide component via the other end of the ADH linker on the nanocarrier to form the corresponding VLP or liposomal peptide complex. Generate.

See Hermanson GT "Bioconjugate Techniques", 2nd Edition Published by Academic Press, Inc., 2008 for a detailed description of the available conjugation methods. In addition to covalent attachment, the components may be attached by adsorption to a preformed synthetic nanocarrier or by encapsulation during synthetic nanocarrier formation.

Any of the immunosuppressive agents provided herein can be used in the methods or compositions provided and, in some embodiments, may be attached to a synthetic nanocarrier. Immunosuppressants include statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histon deacetylase (HDAC) inhibitors; corticosteroids; Inhibitors; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors; proteasome inhibitors; kinase inhibitors; G protein-conjugated receptor agonists; G protein-conjugated receptor antagonists; glucos Corticoids; Retinoids; Cytokine Inhibitors; Cytokine Receptor Inhibitors; Cytokine Receptor Activators; Peroxysome Proliferator-Activated Receptor Agonists; Peroxysome Proliferator-Activated Receptor Agonists; Histon Deacetylase Inhibitors; However, it is not limited to these. Immunosuppressants also include IDO, vitamin D3, cyclosporine A, arylhydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, triporide, interleukin (eg IL-1). , IL-10), cyclosporin A, cytokines or siRNAs that target cytokine receptors, and the like.

Examples of statins include atorvastatin (LIPITOR®, TORVAST®), simvastatin, fulvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®, ALTOCOR (registered trademark)). Registered trademark), ALTOPREV (registered trademark)), mevastatin (COMPACTIN (registered trademark)), pitavastatin (LIVALO (registered trademark), PIAVA (registered trademark)), losuvastatin (PRAVACHOL (registered trademark), SELEKTINE (registered trademark), LIPOSTAT (Registered Trademarks)), losuvastatin (CRESTOR®) and simvastatin (ZOCOR®, LIPEX®).

Examples of mTOR inhibitors include rapamycin and its relatives (eg, CCL-779, RAD001, AP23573, C20-metallyl rapamycin (C20-Marap), C16- (S) -butylsulfonamide rapamycin (C16-BSrap), C16- (S) -3-methylindol rapamycin (C16-iRap) (Bayle et al., Chemistry & Biology 2006, 13: 99-107), AZD8055, BEZ235 (NVP-BEZ235), chrysophanoic acid (crisofa) Noor), Defololimus (MK-8669), Everolimus (RAD0001), KU-0063794, PI-103, PP242, Temsirolimus and WYE-354 (available from Selleck, Houston, TX, USA).

Examples of TGF-β signaling agents include TGF-β ligands (eg, activin A, GDF1, GDF11, bone morphogenic proteins, nodal, TGF-β) and their receptors (eg, ACVR1B, ACVR1C, ACVR2A, ACVR2B). , BMPR2, BMPR1A, BMPR1B, TGFβRI, TGFβRII), R-SMADS / co-SMADS (eg SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8) and ligand inhibitors (eg, follistatin, nogin, cordin, DAN, lefty) , LTBP1, THBS1, decorin).

Examples of inhibitors of mitochondrial function include atractyroside (dipotassium salt), valinomycin acid (triammonium salt), carbonyl cyanide m-chlorophenylhydrazone, caroboxyattractyroside (eg from the genus Attractiris), CGP -37157, (-)-deguelin (eg from Mundulea sericea), F16, hexokinase IIVDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1 and valinomycin (eg from Streptomuces fulvissimus) (EMD4Biosciences, USA).

Examples of P38 inhibitors are SB-203580 (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) 1H-imidazole), SB-239063 (trans-1- (4-Hydroxycyclohexyl) -4- (fluorophenyl) -5- (2-methoxy-pyrimidine-4-yl) imidazole), SB-220025 (5- (2amino-4-pyrimidinyl) -4- (4-fluoro) Phenyl) -1- (4-piperidinyl) imidazole)) and ARRY-797.

Examples of NF (eg, ΝΚ-κβ) inhibitors include IFRD1, 2- (1,8-naphthylidine-2-yl) -phenol, 5-aminosalicylic acid, BAY11-7082, BAY11-7085, CAPE (caffeic acid). Phenol ester), diethyl maleate, IKK-2 inhibitor IV, IMD0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB activation inhibitor III, NF-κB activation inhibitor II, JSH- 23, Parthenolide, phenylalcin oxide (PAO), PPM-18, pyrrolidinedithiocarbamate ammonium salt, QNZ, RO106-9920, locagrammid, locagrammid AL, locagrammid C, locagramid I, locagramid J, locagramol, (R) -MG-132 , Sodium salicylate, triptolide (PG490) and wederolactone.

Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.
Examples of prostaglandin E2 agonists include E-prostanoid 2 and E-prostanoid 4.
Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) include caffeine, aminophyllin, IBMX (3-isobutyl-1-methylxanthin), paraxanthin, pentoxyfilin, theobromine, theophylline, methylated xanthin, vardenafil, EHNA (Erythro-9- (2-hydroxy-3-nonyl) adenine), anagrelide, enoxymon (PERFAN ™), milllinone, levosimendon, mesembrin, ibudilast, picramirast, luteolin, drotaverin, roflumilast (DAXAS) ), DALIRESP (trademark)), sildenafil (REVATION (registered trademark), VIAGRA (registered trademark)), tadalafil (ADCIRCA (registered trademark), CIALIS (registered trademark)), vardenafil (LEVITRA (registered trademark), STAXYN (registered trademark) )), Udenafil, Avanafil, Icarine, 4-methylpiperazine and pyrazolopyrimidine-7-1.

Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate and salinosporamide A.
Examples of kinase inhibitors are bevasizumab, BIBW2992, cetuximab (ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib (IRESSA®), ranibizumab (LUCENTIS). (Registered Trademarks)), pegaptanib, sorafenib, dasatinib, snitinib, elrotinib, nirotinib, ranibizumab, panitummab, bandetanib, E7080, pazopanib and mubritinib.

Examples of glucocorticoids include hydrocortisone (cortisol), cortisol acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, bechrometasone, fludrocortisone acetate, deoxycortisone acetate (DOCA) and aldosterone.
Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®, SOTRET®). ), Aritretinoin (PANRETIN®), Etretinoin (TEGISON®) and its metabolites acitretin (SORIATANE®), Tazarotene (TAZORAC®, AVAGE®, ZORAC® )), Bexarotene (TARGRETIN®) and adaparene (DIFFERIN®).

Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonists, IGFBP, TNF-BF, uromodulin, alpha-2-macroglobulin, cyclosporin A, pentamidine and pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®) can be mentioned.
Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARγ antagonist III, G335 and T0070907 (EMD4Biosciences, USA).
Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY171883, PPARγ activator, Fmoc-Leu, troglitazone and WY-14643 (EMD4Biosciences, USA). Can be mentioned.

Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamic acids) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B) and depsipeptides, fats such as benzamides, electrophilic ketones, phenyl butyrate and valproic acid. Derivatives of group acid compounds, hydroxamic acids such as vorinostat (SAHA), verinostat (PXD101), LAQ824 and panobinostat (LBH589), benzamides such as entinostat (MS-275), CI994 and mosetinostat (MGCD0103), nicotinamide, NAD. , Dihydrocoumarin, naphthopyranone and 2-hydroxynaphaldehyde.

Examples of calcineurin inhibitors include cyclosporin, pimecrolimus, voclosporin and tacrolimus.
Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyculin A, okadaic acid, cantalidine, cypermesulin, ethyl-3,4-dehostatin, phosphatase sodium salt, MAZ51, methyl-3,4-dehostatin, NSC95397. , Norcantarisine, ammonium okadaate from prorocentrum concavum, okadaic acid, potassium okadaate, sodium okadaate, phenylalsin oxide, various phosphatase inhibitor mixed solutions, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1 , Protein phosphatase 2A2 and sodium orthovanadate.

In some aspect of any one of the methods, compositions or kits provided, the therapeutic macromolecules described herein are also attached to synthetic nanocarriers. In other embodiments, the therapeutic macromolecule is not attached to any synthetic nanocarrier. In some aspects of any of these situations, the therapeutic macromolecule may be delivered in the form of the therapeutic macromolecule itself or a fragment or derivative thereof.

Therapeutic macromolecules may include therapeutic proteins or therapeutic polynucleotides. Therapeutic proteins include instillable therapeutic proteins, enzymes, enzyme cofactors, hormones, blood coagulation factors, cytokines and interferons, growth factors, monoclonal antibodies and polyclonal antibodies (eg, those administered to the subject as replacement therapy). And proteins associated with Pompe's disease such as, but not limited to, acidic glucosidase alpha, rhGAA (eg, myozyme and luminzyme). Therapeutic proteins also include proteins involved in the blood coagulation cascade. Therapeutic proteins include Factor VIII, Factor VII, Factor IX, Factor V, von Villebrand Factor, von Heldebrandt, Tissue Plasminogen Activator, Insulin, Growth Hormone, Erythropoetin Alpha, VEGF, Examples include, but are not limited to, thrombopoetin, lysoteam, antithrombin, etc. Therapeutic proteins also include adipokines such as leptin and adiponectin. Other examples of therapeutic proteins are described below and are described herein. As described separately inside.

Examples of therapeutic proteins used in enzyme replacement therapy for subjects with lysosomal storage disorders include imiglucerase for the treatment of Gauche disease (eg CEREZYME ™) and a-galactosidase A for the treatment of Fabry disease a-galA) (eg, agarsidase beta, FABRYZYME ™), acidic α-glucosidase (GAA) for the treatment of Pompe disease (eg, acidic glucosidase alpha, LUMIZYME ™, MYOZYME ™), mucopolysaccharides Arylsulfase B for the treatment of disease (eg, laronidase, ALDURAZYME ™, idursulfase, ELAPRASE ™, arylsulfase B, NAGLAZYME ™), peglotycase (KRYSTEXXA) and pegushiticase. , Not limited to these.

Examples of enzymes include oxidoreductase, transferase, hydrolase, lyase, isomerase, asparaginase, uricase, glycosidase, asparaginase, uricase, protease, nuclease, collagenase, hyaluronidase, heparinase, heparanase, lyase and ligase.
Therapeutic proteins may also include enzymes, toxins, or other proteins or peptides isolated or derived from any bacterial, fungal, or viral source. ..

Examples of hormones include melatonin (N-acetyl-5-methoxytryptamine), serotonin, tyrosin (or tetraiodotyronin) (thyroid hormone), triiodotyronin (thyroid hormone), epinephrine (or adrenaline), Norepinephrine (or noradrenaline), dopamine (or prolactin inhibitor hormone), anti-Muller tube hormone (or Muller tube inhibitor or hormone), adiponectin, corticotropin (or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or diuretic hormone) Or vasopresin, arginine vasopresin), atrial sodium diuretic peptide (or atriopeptin), calcitonin, cholesistkinin, corticotropin-releasing hormone, erythropoetin, follicular stimulating hormone, gastrin, grelin, glucagon, glucagon-like peptide (GLP-1) GIP, gonadotropin-releasing hormone, growth hormone-releasing hormone,

Human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (or somatomedin), leptin, luteinizing hormone, melanin cell stimulating hormone, olexin, oxytocin, parathyroid hormone, prolactin, reluxin, secretine , Somatostatin, thrombopoetin, thyroid stimulating hormone (or tyrotropin), tyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstendione, dihydrotestosterone, estradiol, estron, estriol, progesterone, calcitriol (1,25) -Dihydroxyvitamin D3), calcidiol (25-hydroxyvitamin D3), prostaglandin, leukotriol, prostacycline, thromboxane, prolactin-releasing hormone, lipotropin, cerebral sodium diuretic peptide, neuropeptide Y, histamine, endoserine, pancreatic polypeptide , Lenin and Enkefarin.

Examples of blood or blood coagulation factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proacceleline, instability), factor VII (stabilizer or proconvertin). ), Factor VIII (antihemopathic globulin), Factor IX (Christmas factor or plasma thromboplastin component), Factor X (Stuart-Prower factor), Factor Xa, Factor XI, Factor XII (Hagemann) Factor), Factor XIII (Fibrin Stabilizer), von Villebrand Factor, Precaricrane (Fletcher Factor), High Polymer Kininogen (HMWK) (Fitzgerald Factor), Fibronectin, Fibrin, Trombin, Antithrombin III, Heparin Assistance Factor II, Protein C, Protein S, Protein Z, Protein Z-related protease inhibitor (ZPI), plasminogen, alpha2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor- 1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagrant or epoetin alfa (Epogen, Procrit).
Examples of cytokines include type 1 cytokines such as lymphokines, interleukins and chemokines, IFN-γ, and type 2 cytokines such as TGF-β and IL-4, IL-10 and IL-13.

Examples of growth factors include adrenomedulin (AM), angiopoetin (Ang), self-secreting cell motility stimulator, bone-forming protein (BMP), brain-derived neurotrophic factor (BDNF), epithelial growth factor (EGF), erythropoetin ( EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony stimulator (G-CSF), granulocyte macrophage colony stimulator (GM-CSF), proliferation differentiation factor- 9 (GDF9), hepatocellular growth factor (HGF), liver cancer-derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulator, myostatin (GDF-8), nerve growth factor (NGF) and others Neurotrophin, platelet-derived growth factor (PDGF), thrombopoetin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), Vascular Endothelial Growth Factor (VEGF), Wnt Signal Path, Placement Growth Factor (P1GF), (Bovine Fetal Somatotropin) (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6 and IL-7 are mentioned.

Examples of monoclonal antibodies include avagobomab, abusiximab, adalimumab, adecatumab, aferimomab, aftuzumab, alacizumab pegol, ALD, alemtuzumab, altumomab pentetate, altumomab pentetate. Anatumomab mafenatox), anlucinzumab, anti-chest gland cytoglobulin, apolizumab, arcitsumomab, aselizumab, atlizumab (tocilizumab), atorolimumab, atorolimumab, bapineozumab, bavituximab, bavituximab , Bisiromab, Bivatuzumab mertansine, Brinatumomab, Brentuximab vedotin, Briaquinumab, Kanaquinumab, Kantuzumab mertansine, Capromab pendetide, Katsumakisomab, Cedelizumab, Sertrizumab , Citatuzumab bogatox, sixtumumab, clenoriximab, kribatsuzumab tetraxetan, konatsumumab, dasetsumab, dacrizumab, daratsumumab, denosumab, detumomab, dollimomab omab , Ecromeximab, Ecromeximab, Ecrizumab, Edbacomab, Edrecolomab, Efarizumab, Efungumab, Erotuzumab, Elsilimomab, Elsilimomab, Enrimomab Pegor, Epitsumomab , Etalashizumab, Exbivirumab,

Fanoresomab, Faralimomab, Faralimomab, Felvizumab, Fezakinumab, Figitumumab, Fontrizumab, Foravirumab, Foravirumab, Foravirumab, Foravirumab, Fresolimumab, Fresolimumab, Fresolimumab Glembatumumab vedotin, Glembatumumab vedotin, Golimumab, Golimiximab, Ibarizumab, Igovomab, Igovomab, Igovomab, Imsyromab, Infliximab, Infliximab Ozogamycin, ipilimumab, iratumumab, keliximab, labetzumab, rebrikizumab, lemalesomab, lelderimumab, lexatumumab, livibirumab (Libivirumab), linzzumab, lintumab, lumiliximab, lumiliximab, lumiliximab, lumiliximab, lumiliximab ), Matsuzumab, Mepolizumab, Metelimumab, Miratuzumab, Minretumomab, Mitsumomab, Mororimumab, Motabizumab, Muromonab-CD3, Nacolomab tafenatox (Nacolomab tafenatox) Nevakumab, Nesitumumab, Nerelimomab, Nimotsuzumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab, Ofatumumab, Ofatumumab, Olaratumab , Ofatumumab, Pascolizumab, Pascolizumab, Pemtumomab, Peltzzumab, Pexerizumab,

Pintumomab, Prilimumab, Pritumumab, Rafivirumab, Ramsilumab, Ranibizumab, Laxibakumab, Regabilumab, Lesrizumab, Lirotsumumab, Lesrizumab, Lirotumumab, Rituximab, Robatumab ), Sevirumab, Sibrotuzumab, Cifalimumab, Siltuximab, Siprizumab, Solanezumab, Sonepcizumab, Sontuzumab, Sontuzumab, Sontuzumab, Stamulumab, , Taplitumomab paptox, Teplizumab paptox, Teplizumab, Telimomab aritox, Tenatumomab, Teneriximab, Teplizumab, Chisilimumab (Tremerizumab), Tocilizumab, Tocilizumab , Tsukotsuzumab Sermoleukin, Tuvirumab, Ultoxazumab, Ustekinumab, Vapaliximab, Bedrizumab, Bertuzumab, Beparimomab, Bisirizumab, Boroshiximab, Bisirizumab, Boroshiximab ). Monoclonal antibodies further include anti-TNF-α antibodies.

Examples of instillable or injectable therapeutic proteins include, for example, tosilitumab (Roche / Actemra®), alpha-1 antitrypsin (Kamada / AAT), hematide® (Affymax and Takeda, synthetic peptides). , Arbuinterferon Alpha-2b (Novartis / Zalbin ™), Lucin® (Pharming Group, C1 inhibitor replacement therapy), Theratechnologies / Egrifta, Synthetic Growth Hormone Release Factor, Oclerisumab (Genentech, Roche and Biogen) ), Berimbab (GlaxoSmithKline / Benlysta®), Pegloticase (Savient Pharmaceuticals / Krystexxa®), Pegloticase, Taligulcerase Alpha (Protalix / Uplyso), Agarcidase Alpha (Shire / Replagal®), Pegloticase alpha (Shire) and keyhole limpet hemocyanin (KLH).

Additional therapeutic proteins include, for example, genetic manipulation proteins such as Fc fusion proteins, bispecific antibodies, multispecific antibodies, nanobodies, antigen binding proteins, antibody fragments and protein complexes such as antibody drug complexes. Be done.

Therapeutic polynucleotides include nucleic acid aptamers such as pegaptanib (McGen, pegged anti-VEGF aptamer), antisense polynucleotides or oligonucleotides (eg, the antiviral drug Fomivirsen or mipomersen, to reduce cholesterol levels. Antisense therapies such as messenger RNA-targeting antisense therapies for aptamer protein B; small interfering RNAs (siRNAs) (eg, 25-30 base pair asymmetry that mediate RNAi with extremely high potency). A double-stranded RNA, a dicer substrate siRNA molecule (DsiRNA); or in US patent application 2013/0115272 (Fougerolles et al.) And published US patent application 2012/0251618 (Schrum et al.). Modified messenger RNAs (mmRNAs) such as those disclosed include, but are not limited to.

Additional therapeutic macromolecules useful according to aspects of the invention will be apparent to those of skill in the art and the invention is not limited in this regard.

In some embodiments, components such as therapeutic macromolecules or immunosuppressants may be isolated. "Isolated" is an element that is isolated from its natural environment and is present in sufficient quantity to allow its identification or use. This means, for example, that the element can be (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. The isolated element may be substantially pure, but does not have to be pure. The elements may be included in the preparation in very small weight percentages, as the isolated elements may be miscible with pharmaceutically acceptable excipients in the pharmaceutical preparation. The elements are nonetheless isolated in that they have been isolated from substances that may be associated in the biological system, i.e. isolated from other lipids or proteins. Any of the elements provided herein may be isolated and included in the composition or used in the method in isolated form.

D. Methods of Making and Using Compositions and Related Methods Some aspects of the invention relate to determining protocols for the methods of concomitant administration provided herein. The protocol can be determined by changing the frequency, dose and other aspects of administration of therapeutic macromolecules and immunosuppressive agents and then assessing the pharmacodynamic effects based on such changes. Modified administration occurs in the presence of an anti-therapeutic macromolecular antibody response. The preferred protocol for the practice of the present invention induces the desired pharmacodynamic effect, but little or no anti-therapeutic polymeric antibody response.

In some aspects of the invention, the desired pharmacodynamic effects of therapeutic macromolecules include stimulating or inhibiting a specific response. In some embodiments, the medicinal effect is, but is not limited to, the production or degradation of cytokines, chemokines, signaling molecules or other molecules; inducing proliferation or death of a particular cell type; a particular cell type. Maturation or localization; interaction with enzymes, structural proteins, carrier proteins or receptor proteins; modulation of activity of enzymes, structural proteins or receptor proteins, etc. In some embodiments, the pharmacodynamic effect is to reduce the production of cytokines, such as inflammation-related cytokines such as TNF, IL-1. In some embodiments, the pharmacodynamic effect is to reduce the activity of cytokines. In some embodiments, the pharmacodynamic effect is to reduce the production of unwanted molecules. In some embodiments, the pharmacodynamic effect is to increase the degradation of unwanted molecules, such as uric acid crystals. In some embodiments, the pharmacodynamic effect is the activity of the enzyme.

The pharmacodynamic effects of therapeutic macromolecules can be assessed by standard methods. In some aspects of the invention, the pharmacodynamic effect is reduction of inflammation. The level of inflammation is not limited, but the following exemplary methods: scoring inflammatory signs such as redness and swelling; scoring joint signs such as mobility, pain or joint destruction; swelling, blood pressure, shortness of breath, etc. Scoring of anaphylactic signs; detecting and / or quantifying cell infiltration by histology, immunohistochemistry, flow cytometry; measuring the concentration of proteins or inflammation-related cytokines such as TNF, IL-1 by ELISA, The expression of a gene or inflammation-related gene can be assessed by transcriptional analysis; measuring the activity of inflammation-related cytokines, and so on.

In some aspects of the invention, the pharmacodynamic effect is the reduction or degradation of unwanted molecules. In some embodiments, the pharmacodynamic effect can be assessed by quantifying, but not limited to, unwanted molecules in tissues or blood samples by methods such as ELISA. In some embodiments, the pharmacodynamic effect can be assessed by quantifying the molecules produced by the degradation of unwanted molecules, such as by ELISA. In some aspects of the invention, the pharmacodynamic effect is the activity of an enzyme that was not previously present or was not properly present. In some embodiments, the activity of the enzyme can be assessed by detecting the presence or concentration of a product of the enzyme activity.

In some aspects of the invention, reduced doses of therapeutic macromolecules are administered to produce pharmacodynamic effects. A reduced dose of therapeutic polymer for this purpose is any of the therapeutic polymers that achieve a pharmacodynamic effect in the presence of an anti-therapeutic polymer antibody response by co-administration of an immunosuppressant dose. Than the dose required to achieve a similar pharmacodynamic effect with a therapeutic polymer when not co-administered with an immunosuppressant dose in the presence of an anti-therapeutic polymer antibody response. Includes small doses. Reduced doses were determined by administering therapeutic macromolecules at specific doses in the presence of anti-therapeutic macromolecular antibody responses, along with immunosuppressive doses, and assessing the pharmacodynamic effects. obtain. The pharmacodynamic effect may then be compared to the pharmacodynamic effect achieved through administration of the therapeutic macromolecule in the presence of an anti-therapeutic macromolecular antibody response without the combined administration of immunosuppressive drug doses. The smaller doses that achieve similar pharmacodynamic effects, as determined by such comparison, are the reduced doses.

As previously mentioned, immunosuppressants may be attached to synthetic nanocarriers. Synthetic nanocarriers can be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers are nanoprecipitated, flow focusing using fluid channels, spray drying, single and double emulsion solvent distillation, solvent extraction, phase separation, milling, microemulsion procedures, micromachining, nanomachining, sacrificial layers, simple. And can be formed by methods such as composite coagulation and other methods well known to those skilled in the art. Alternatively or additionally, aqueous and organic solvent synthesis for monodisperse semiconductors or conductive, magnetic, organic and other nanomaterials has been described (Pellegrino et al., 2005, Small, 1:48). Murray et al., 2000, Ann. Rev. Mat. Sci., 30: 545; Trindade et al., 2001, Chem. Mat., 13: 3843). Additional methods are described in the literature (eg Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5 : 13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35: 755; US Pat. Nos. 5,587,325 and 6007845; P. Paolicelli et al. ., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5 (6): 843-853 (2010)).

If necessary, C. Astete et al., "Synthesis and characterization of PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis " Pegylated Poly (Lactide) and Poly (Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery "Current Drug Delivery 1: 321-333 (2004); C. Reis et al.," Nanoencapsulation I. Methods for preparation of drug-loaded polypropylene nanoparticles "Nanomedicine 2: 8-21 (2006); P. Paolicelli et al.," Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles "Nanomedicine. 5 (6) ): 843-853 (2010), but various methods may be used to encapsulate various materials in synthetic nanocarriers. Use other methods, including, but not limited to, methods disclosed in US Pat. No. 6,632,671 (Unger, issued October 14, 2003), which are suitable for encapsulating materials in synthetic nanocarriers. You may.

In certain embodiments, synthetic nanocarriers are prepared by nanoprecipitation process or spray drying. The conditions used in the preparation of synthetic nanocarriers can be changed to produce particles of the desired size or properties (eg, hydrophobicity, hydrophilicity, external morphology, "stickiness", shape, etc.). The method of preparation of the synthetic nanocarrier and the conditions in which it is used (eg, solvent, temperature, concentration, air flow rate, etc.) may depend on the composition of the material and / or polymer matrix to adhere to the synthetic nanocarrier.

If the synthetic nanocarriers prepared by any of the above methods have a size range outside the desired range, such synthetic nanocarriers may be granulated, for example, using a sieve.
The elements (ie, components) of the synthetic nanocarrier (ie, components, antigens, immunosuppressants, etc.) may be attached to the entire surface of the synthetic nanocarrier by, for example, one or more covalent bonds, or one or two. It may be attached using one or more linkers.

Additional methods for functionalizing synthetic nanocarriers are published in US Patent Application 2006/0002852 (Saltzman et al.), Published US Patent Application 2009/0028910 (DeSimone et al) or published. It may be adapted from international patent application WO / 2008/127532Al (Murthy et al.).

Alternatively or additionally, the synthetic nanocarrier may be attached to the constituents, either directly or indirectly through non-covalent interactions. In non-covalent embodiments, non-covalent attachment is charge interaction, affinity interaction, metal coordination, physsisorption, host-guest interaction, hydrophobic interaction, TT stacking interaction, hydrogen bond interaction. Mediated by non-covalent interactions, including, but not limited to, actions, van der Waals interactions, magnetic interactions, electrostatic interactions, bipolar-dipole interactions and / or combinations thereof. .. Such linkages may be arranged on the outer or inner surface of the synthetic nanocarrier. In some embodiments, encapsulation and / or absorption is a form of attachment. In some embodiments, the synthetic nanocarriers may be combined with therapeutic macromolecules or other compositions by mixing in the same vehicle or delivery system.

The compositions provided herein include inorganic or organic buffers (eg, sodium or potassium salts of phosphoric acid, carboxylic acid, acetic acid or citric acid) and pH adjusters (eg, hydrochloric acid, sodium hydroxide or potassium). Citric acid or acetic acid salts, amino acids and salts thereof), antioxidants (eg ascorbic acid, alpha-tocopherol), surfactants (eg polysolvate 20, polysolvate 80, polyoxyethylene 9-10 nonylphenol, deoxychol Sodium acid), lysed and / or frozen / lyo stabilizers (eg, sucrose, lactose, mannitol, trehalose), osmotic regulators (eg, salts or sugars), antibacterial agents (eg, benzo) Acids, phenols, gentamycins), antifoaming agents (eg, polydimethylsilozone), preservatives (eg, timerosar, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (eg, polyvinylpyrrolidone, poroxamer). 488, carboxymethyl cellulose) and co-solvents (eg, glycerol, polyethylene glycol, ethanol) may be included.

Compositions according to the invention may contain pharmaceutically acceptable excipients. The composition can be made using conventional pharmaceutical manufacturing and compounding techniques to reach a useful dosage form. Suitable techniques for use in the practice of the present invention are the Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc .; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by ME Auten, 2001, Churchill Livingstone. In some embodiments, the composition is suspended in sterile aqueous saline solution for injection with a preservative.

It should be understood that the compositions of the present invention can be made in any suitable manner and the present invention is not limited to compositions which can be produced using the methods described herein. Choosing the appropriate manufacturing method may require attention to the nature of the specific parts involved.

In some embodiments, the composition is manufactured under sterile conditions or is finally sterile. This can ensure that the resulting composition is sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides valuable safety measures, especially when the subject receiving the composition has an immune deficiency, suffers from an infection, and / or is susceptible to infection. In some embodiments, the composition may be lyophilized and stored for extended periods of time without loss of activity, depending on the formulation strategy, in suspension or as a lyophilized powder.

Administration according to the invention includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, transmucosal, transdermal, transcutaneous or intradermal routes. In a preferred embodiment, administration is via subcutaneous route administration. The compositions referred to herein can be prepared and prepared for administration, preferably combination administration, using conventional methods.

The composition of the present invention can be administered in an effective amount, such as the effective amount described separately herein. Dosage form doses may contain varying amounts of immunosuppressive agents and / or therapeutic macromolecules in accordance with the present invention. The amount of immunosuppressive and / or therapeutic macromolecule present in the dosage form of the invention is modified according to the nature of the therapeutic macromolecule and / or immunosuppressant, the therapeutic effect to be performed and other such parameters. obtain. In some embodiments, dose range studies may be performed to establish optimal therapeutic doses of immunosuppressive agents and / or therapeutic macromolecules that should be present in the dosage form.

In some embodiments, the immunosuppressant and / or therapeutic macromolecule is an amount effective to generate a reduced immune response to the desired pharmacodynamic effect and / or therapeutic macromolecule when administered to the subject. It is present in the dosage form. It may be possible to use conventional dose range studies and techniques in the subject to determine the amount of immunosuppressant and / or therapeutic macromolecule effective in achieving the desired results. The dosage forms of the invention can be administered at varying frequencies. In a preferred embodiment, at least one dose of the composition provided herein is sufficient to generate a pharmacologically valid response. In a more preferred embodiment, at least two or at least three doses are utilized to ensure a pharmacologically valid response. In some embodiments, repeated administrations are utilized to ensure a pharmacologically valid response.

Another aspect of this disclosure relates to kits. In some embodiments, the kit comprises a pharmacodynamically effective dose of the therapeutic macromolecule, such as a reduced pharmacodynamically effective dose, or a dose greater than one. In such an embodiment, the kit may also contain an immunosuppressant dose or a dose greater than one of the immunosuppressants. Immunosuppressant doses and pharmacodynamically effective doses may be contained in separate containers or in the same container in the kit. In some embodiments, the container is a vial or ampoule. In some embodiments, the pharmacodynamically effective dose and / or immunosuppressive drug dose of the Therapeutic Polymer is contained in a solution separate from the container and the pharmacodynamically effective dose and / or of the Therapeutic Polymer. The immunosuppressant dose may be adapted to be added to the vessel at a later time.

In some embodiments, the pharmacodynamically effective dose and / or immunosuppressant dose of the Therapeutic Polymer is in a lyophilized form, respectively, in a separate container or in the same container, which at a later time. It may be reconstructed. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments, the instructions for use include a description of the methods described herein. The instructions for use may be in any suitable form, for example a printed insert or label. In some embodiments, the kit further comprises one or more syringes.

Example
Example 1: Evaluation of immune response by synthetic nanocarriers containing immunosuppressants and APC presentable antigens in vivo 17 amino acid peptides known to be T and B cell epitopes of ovoalbumin protein. , Ovoalbumin peptides _323-339 (OVA 323-339), were purchased from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505) (Part # 4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (Product Catalog # R1017). A PLGA with a lactide: glycolide ratio of 3: 1 and an intrinsic viscosity of 0.75 dL / g was purchased from Sur Modics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

Method for preparing synthetic nanocarriers containing rapamycin and ovalbumin (323-339) The solution was prepared as follows:
Solution 1: OVA _323-339 , 20 mg / mL in dilute aqueous hydrochloric acid solution. This solution was prepared by dissolving the ovalbumin peptide in 0.13M hydrochloric acid solution at room temperature.
Solution 2: PLGA, 100 mg / mL in methylene chloride. This solution was prepared by dissolving PLGA in pure methylene chloride.
Solution 3: PLA-PEG, 100 mg / mL in methylene chloride. This solution was prepared by dissolving PLA-PEG in pure methylene chloride.
Solution 4: rapamycin, 50 mg / mL in methylene chloride. This solution was prepared by dissolving rapamycin in pure methylene chloride.
Solution 5: polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

First, a water-in-oil emulsion was prepared. Combine Solution 1 (0.2 mL), Solution 2 (0.75 mL), Solution 3 (0.25 mL) and Solution 4 (0.2 mL) in a small pressure tube and 50% amplitude using Branson Digital Sonifier 250. W1 / O1 was prepared by sonication for 40 seconds. Solution 5 (3.0 mL) was then combined with the primary W1 / O1 emulsion, vortexed for 10 s, and sonicated with a Branson Digital Sonifier 250 at 30% amplitude for 60 seconds to give the secondary emulsion (W1 / O1 /). W2) was prepared.

The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate methylene chloride to form synthetic nanocarriers. By transferring the synthetic nanocarrier suspension to a centrifuge tube and centrifuging at 75,600 xg and 4 ° C. for 35 min, removing the supernatant and resuspending the pellet in phosphate buffered saline. A portion of the synthetic nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final synthetic nanocarrier dispersion of about 10 mg / mL.
The amounts of peptide and rapamycin in the synthetic nanocarrier were determined by HPLC analysis. The total dry synthetic nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Methods for Synthetic Nanocarriers Containing Rapamycin First, a water-in-oil emulsion was prepared. Combine 0.13M hydrochloric acid solution (0.2 mL), solution 2 (0.75 mL), solution 3 (0.25 mL) and solution 4 (0.2 mL) in a small pressure tube and use the Branson Digital Sonifier 250. W1 / O1 was prepared by ultrasonic treatment at 50% amplitude for 40 seconds. The secondary emulsion (W1 / O1) was then combined with solution 5 (3.0 mL), vortexed for 10 s and sonicated with a Branson Digital Sonifier 250 at 30% amplitude for 60 seconds. / W2) was prepared.

The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate methylene chloride to form synthetic nanocarriers. By transferring the synthetic nanocarrier suspension to a centrifuge tube and centrifuging at 21,000 xg and 4 ° C. for 1 hour, removing the supernatant and resuspending the pellet in phosphate buffered saline. , A part of the synthetic nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final synthetic nanocarrier dispersion of about 10 mg / mL.

The amount of rapamycin in the synthetic nanocarrier was determined by HPLC analysis. The total dry synthetic nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Method for measuring rapamycin loading capacity Approximately 3 mg of synthetic nanocarriers were collected and centrifuged to separate the supernatant from the synthetic nanocarrier pellets. Acetonitrile was added to the pellet and the sample was sonicated and centrifuged to remove any insoluble material. The supernatant and pellet were injected into RP-HPLC and the absorbance at 278 nm was read. The μg found in the pellet was used to calculate the captured% (load), and the supernatant and μg in the pellet were used to calculate the total μg recovered.

Method for Measuring Ovalbumin (323-339) Load Approximately 3 mg of synthetic nanocarriers was collected and centrifuged to separate the supernatant from the synthetic nanocarrier pellets. Trifluoroethanol was added to the pellet and the sample was sonicated to dissolve the polymer, 0.2% trifluoroacetic acid was added to sonicate the sample, and then centrifugation was performed to remove any insoluble material. The supernatant and pellet were injected into RP-HPLC and the absorption at 215 nm was read. The μg found in the pellet was used to calculate the captured% (load) and the supernatant and μg in the pellet were used to calculate the total μg recovered.

Immunization The purpose of this experiment is to assess the effect of encapsulated immunosuppressants on an ongoing antibody response by measuring antigen-specific immunoglobulins. One group of animals was left unimmunized as a control. Two groups of animals were immunized with chicken ovalbumin (OVA) and CpG by three injections in the footpad (d0, d14 and d28), followed by antibody titers. For immunization, the animals were s. c. Received 20 μl / limb OVA + CpG (12.5 μg OVA + 10 μg CpG). Treatments administered on the same day include i. v. Administration (200 μl) or s. c. Administration (20 μl) was included. The nanocarriers were diluted by injecting the same amount of OVA _{323-339 into the treatment group.}

Measurement of IgG The level of IgG antibody was measured. Blocker Casein in PBS (Thermo Fisher, Catalog # 37528) was used as the diluent. 10 mL Tween-20 (Sigma, Catalog # P9416-100 mL), 2 liters of 10 x PBS stock (PBS: OmniPur® 10X PBS Liquid Concentrate, 4L, EMD Chemicals, Catalog # 6505) and 18 liters of deionization 0.05% Tween-20 in PBS, prepared by addition to water, was used as a wash buffer.
An OVA protein with a stock concentration of 5 mg / mL was used as a coating material. It was diluted 1: 1000 to 5 μg / ml and used as the concentration used. Each well of the assay plate was coated with 100 μl of diluted OVA per well, the plate was sealed with a sealing membrane (VWR, Catalog # 60941-120) and incubated overnight at 4 ° C. A 96-well flat bottom plate of Costar 9017 was used as the assay plate (Costar 9017).

A low-binding polypropylene 96-well plate or tube was used as the setup plate and samples were prepared in it prior to transfer to the assay plate. The setup plate did not contain any antigen and therefore serum antibodies did not bind to the plate during sample setup. When the setup plate was used for sample preparation and the sample was prepared using an antigen-coated plate, the binding that would occur during sample preparation or pipetting was minimized. Prior to preparing samples in setup plates, wells were covered with diluent to block any non-specific binding, the plates were sealed and incubated overnight at 4 ° C.

The assay plate was washed 3 times with wash buffer and the wash buffer was completely aspirated from the wells after the final wash. After washing, 300 μl of diluent was added to each well of the assay plate (s) to block non-specific binding and the plates were incubated at room temperature for at least 2 hours. Serum samples were prepared in setup plates with appropriate starting dilutions. In some cases, starting dilutions were prepared using diluents in 1.5 mL tubes and then transferred to setup plates. Appropriate starting dilutions, if available, were determined based on prior data. If no prior data was available, the lowest starting dilution was 1:40. Once diluted, 200 μl of the starting dilution serum sample was transferred from the tube to the appropriate well of the setup plate.

An exemplary setup plate layout is described below. Rows 2 and 3 contained an anti-ovalbumin monoclonal IgG2b isotype (AbCam, ab17291) standard diluted to 0.25 μg / ml (1: 4000 dilution). Rows 4-11 contained serum samples (appropriate dilutions). Columns 1 and 12 were not used in the sample or standard to avoid any bias in the measurements due to the edge effect. Instead, columns 1 and 12 contained 200 μl of diluent. Normal mouse serum diluted 1:40 was used as a negative control. Anti-mouse IgG2a diluted 1: 500 from a 0.5 mg / mL stock (BD Bioscience) was used as an isotype control.

Once all samples were prepared in the setup plate, the plate was sealed and stored at 4 ° C. until blocking of the assay plate was complete. The assay plate was washed 3 times with wash buffer and the wash buffer was completely aspirated after the final wash. After washing, 100 μl of diluent was added to all wells of rows B to H of the assay plate. Samples were transferred from the setup plate to the assay plate using a 12-channen pipette. Prior to transfer, samples were mixed by pipetting 150 μl of diluted serum up and down three times. After mixing, each 150 μ1 sample was removed from the setup plate and added to row A of each assay plate.

Once the starting dilution of each sample was transferred from the setup plate to row A of the assay plate, the serial dilution was pipetted onto the assay plate as follows. Each 50 μl serum sample was removed from row A using a 12 channel pipette and mixed with 100 μl diluent previously added to each well in row B. This step was repeated downwards throughout the plate. After pipetting the dilution of the last row, 50 μl of fluid was removed from the wells of the last row and discarded, resulting in a final volume of 100 μl per well of the assay plate. After preparing the sample dilution in the assay plate, the plate was incubated at room temperature for at least 2 hours.

After incubation, the plates were washed 3 times with wash buffer. The detection antibody (goat anti-mouse anti-IgG, HRP complex, AbCam ab98717) was diluted 1: 1500 (0.33 μg / ml) with diluent and 100 μl of diluted antibody was added to each well. The plates were incubated at room temperature for 1 hour and then washed 3 times with wash buffer so that each wash step included a soaking time of at least 30 seconds.

After washing, the detection substrate was added to the wells. Equal parts of Substrate A and Substrate B (BD Biosciences TMB Substrate Reagent Set, Catalog # 555214) were combined just prior to addition to the assay plate, 100 μl of mixed substrate solution was added to each well and incubated in the dark for 10 minutes. did. After 10 minutes, the reaction was stopped by adding 50 μl stop solution (2N H ₂ SO _{4) to each well.} The optical density (OD) of the well immediately after the stop solution was added was assessed at 450 nm with a plate reader and subtracted at 570 nm. Data analysis was performed using the Molecular Device software SoftMax Pro v5.4. The dilution was taken on the x-axis (log scale) and the OD value was taken on the y-axis (uniform scale) to prepare a graph that fits the 4-parameter logistic curve, and the maximum half-volume value (EC ₅₀ ) of each sample was determined. The plate template at the top of the layout was adjusted to reflect the dilution of each sample (one per row).

Results Results demonstrate the ability to use immunosuppressive drug doses in combination with antigens to reduce antigen-specific antibody responses when both immunosuppressants and antigens are attached to nanocarriers. FIG. 1 shows a decrease in circulating antigen-specific antibody production by nanocarriers containing peptide antigens and immunosuppressants when administered intravenously. Two independent experiments were performed using 5 animals in each experiment. FIG. 2 shows a decrease in the production of antigen-specific circulating antibody due to a nanocarrier containing a peptide antigen and an immunosuppressant when administered subcutaneously. Three independent experiments were performed using 5 animals in each experiment. The P-value was calculated using the Bonferroni post test of the regular one-way ANOVA test (* = p <0.05, ** = p <0.01 and *** = p <0.001).

These results demonstrate that the same amount of ovalbumin protein can be used to achieve improved effects when administered under conditions that would normally produce a significant anti-ovalbumin antibody response. ing. Moreover, as this result suggests, this pharmacodynamic effect would have been obtained at a reduced pharmacodynamically effective dose. Finally, a protocol has been established that has been demonstrated to produce pharmacodynamic effects at reduced pharmacodynamically effective doses of therapeutic macromolecules when used in combination with immunosuppressive drug doses. Supported by.

Example 2: Evaluation of immune response following concomitant administration of synthetic nanocarriers containing immunosuppressants and therapeutic proteins Material rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (Product Catalog # R1017). PLGA with 76% lactide and 24% glycolide content and intrinsic viscosity of 0.69 dL / g was purchased from Sur Modics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG block copolymers with approximately 5,000 Da PEG blocks and approximately 40,000 Da PLA blocks were purchased from Sur Modics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 5CE). Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

Preparation method of synthetic nanocarrier The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA and 25 mg / mL PLA-PEG in methylene chloride. This solution was prepared by dissolving PLGA and PLA-PEG in pure methylene chloride.
Solution 2: rapamycin, 100 mg / mL in methylene chloride. This solution was prepared by dissolving rapamycin in pure methylene chloride.
Solution 3: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

Nanocarriers were prepared using oil-in-water emulsions. O / by combining solution 1 (1 mL), solution 2 (0.1 mL) and solution 3 (3 mL) in a small pressure tube and sonicating at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. A W emulsion was prepared. This O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate methylene chloride to form nanocarriers. Nano by transferring the nanocarrier suspension to a centrifuge tube and centrifuging at 75,600 xg and 4 ° C. for 35 min, removing the supernatant and resuspending the pellet in phosphate buffered saline. A portion of the carrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final nanocarrier dispersion of about 10 mg / mL.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Response to ovalbumin C57BL / 6 same-age (5-6 weeks) female mice (5 per group, 3 groups; unsensitized controls, untreated controls and treatment with synthetic nanocarriers with rapamycin) immunogenic 25 μg of chicken ovalbumin induced using high shear to form aggregates to increase aggOVA) was intravenously injected. After these intravenous injections (administration on days 0, 14 and 28) that antigen stimulate and boost the immune response, a subsequent boost of 25 μg ovalbumin intraperitoneally (ip, days 42 and 57). A 12.5 μg pOVA challenge (s.c., d62) was performed under the skin of the left hind limb, followed by a challenge (s.c., 90th, 105, and 135 days) using the same aggOVA in combination with CpG. It was. FIG. 3 shows the results of such an immunization protocol. On days 25 and 40, ELISA (generally a technique as described in Example 1) can be used to detect prominent anti-OVA antibody (Ab) responses in these animals.

Subsequent aggOVA injections were observed to maintain these antibody titers or further boost the response. On the other hand, i. v. Mice were treated with the administered synthetic nanocarrier (a dose calculated to provide 100 μg of rapamycin) with aggOVA only during the first three encounters with the antigens d0, 14 and 28. Occasionally, no IgG response can be detected for the next 60 days, even after four injections with an immunogenic mixture containing aggOVA. See FIG. Very slight anti-agOVA IgG responses can be detected in treated animals three times with the highly immunogenic combination aggOVA / KLH and CpG. c. Limited after injection (s.c., 90, 105 and 135 days).

Response to the second antigen, keyhole limpet hemocyanin The same mice as above were also injected with 0.05 μg of the second Ag, keyhole limpet hemocyanin (KLH), but the KLH was mixed with aggOVA. It was administered according to the above schedule of aggOVA. The anti-KLHIgG antibody titer was determined using a method similar to the anti-OVA IgG titer method described in Example 1.

The results in FIG. 4 show that, unlike aggOVA, KLH is not immunogenic at the first stage of its encounter with Ag (d0-40). However, KLH is a three-time s. Of KLH in the presence of CpG. c. After immunization (s.c., 90th, 105th and 135th days), it was immunogenic in animals untreated with synthetic nanocarriers. A strong anti-KLH response can be observed in these control mice, while mice that received three treatments with a synthetic nanocarrier carrying an immunosuppressant in combination with an antigen at the beginning of this protocol were KLH /. It has no detectable response even after a CpG challenge.

These results demonstrate that the compositions and methods provided herein provide improved efficacy when administered under conditions that would normally produce a significant antibody response. There is. These results also support the establishment of protocols that have been demonstrated to produce pharmacodynamic effects.

Example 3: Method for preparing nanocarriers attached to rapamycin The material rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (product code R1017). A PLGA with a lactide: glycolide ratio of 3: 1 and an intrinsic viscosity of 0.75 dL / g was purchased from Sur Modics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride.
Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

Nanocarriers were prepared using oil-in-water (O / W) emulsions. Prepare an O / W emulsion by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. did. The O / W emulsion was added to a beaker containing a 70 mM pH 8 phosphate buffer solution and stirred at room temperature for 2 hours to evaporate methylene chloride to form nanocarriers. The nanocarrier suspension was transferred to a centrifuge tube and centrifuged at 75,600 xg and 4 ° C. for 50 min, the supernatant was removed, and the pellet was resuspended in phosphate buffered saline to resuspend the nanocarrier. Was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final nanocarrier dispersion of about 10 mg / mL.

The size of the nanocarrier was determined by dynamic light scattering and is shown in Table 1. The amount of rapamycin in the nanocarrier was determined by HPLC analysis (Table 1). The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA01702) (product code R1017). PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride.
Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.
Solution 3: 70 mM phosphate buffer, pH 8.

Solution 1 (1.0 mL) and Solution 2 (3.0 mL) are mixed in a small glass pressure tube and sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250 to make an oil-in-water emulsion. did. The emulsion was added to an open 50 mL beaker containing Solution 3 (30 mL) and stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in the suspension. A portion of the suspended nanocarrier is then transferred to a centrifuge tube and rotated at 75,600 rcf for 40 minutes to remove the supernatant and resuspend the pellet in phosphate buffered saline. Was washed. The washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA01702) (product code R1017). PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). The F8II.1723 peptide was purchased from AnaSpec (34801 Campus Drive, Fremont, CA 94555). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows.
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe, and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe, and rapamycin in pure methylene chloride.
Solution 2: 10 mg / mL F8II. In 50 mM pH 11.5 phosphate buffer. 1723 (ERLWDYGMSSSPHVL) and 100 mg / mL sucrose. This solution dissolves sucrose in 50 mM pH 11.5 phosphate buffer, followed by F8II. Prepared by adding 1723 peptide as a dry powder.
Solution 3: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.
Solution 4: 70 mM pH 8 phosphate buffer.

First, solution 1 (1.0 mL) and solution 2 (0.2 mL) are mixed in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250 to primary (W1). / O) An emulsion was prepared.
Solution 3 (3.0 mL) was then added to the primary emulsion and vortexed to make a crude dispersion, which was then sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. A W1 / O / W2) emulsion was formed.

The secondary emulsion was added to an open 50 mL beaker containing Solution 4 (30 mL) and stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in the suspension. The nanocarrier suspension was then transferred to a centrifuge tube and rotated at 75,600 rcf for 35 minutes to remove the supernatant and resuspend the pellet in phosphate buffered saline to allow the suspended nanocarriers. A part was washed. The washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The amount of F8II.1723 in the nanocarrier was determined using a fluorescamine-based assay. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA01702) (product code R1017). PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). The F8II.75 peptide was purchased from AnaSpec (34801 Campus Drive, Fremont, CA 94555). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride.
Solution 2: 10 mg / mL F8II.75 (VHLFNIAKPRPPWMG) and 100 mg / mL sucrose in 50 mM pH 2 phosphate buffer. This solution was prepared by dissolving sucrose in 50 mM pH 2 phosphate buffer and then adding the F8II.1723 peptide as a dry powder.
Solution 3: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.
Solution 4: 70 mM pH 8 phosphate buffer.

First, solution 1 (1.0 mL) and solution 2 (0.2 mL) are mixed in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250 to primary (W1). / O) An emulsion was prepared.
Solution 3 (3.0 mL) was then added to the primary emulsion and vortexed to make a crude dispersion, which was then sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. A W1 / O / W2) emulsion was formed.

The secondary emulsion was added to an open 50 mL beaker containing Solution 4 (30 mL) and stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in the suspension. The nanocarrier suspension was then transferred to a centrifuge tube and rotated at 75,600 rcf for 35 minutes to remove the supernatant and resuspend the pellet in phosphate buffered saline to allow the suspended nanocarriers. A part was washed. This washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The amount of F8II.75 in the nanocarrier was determined using a fluorescamine-based assay. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Material Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA01702) (product code R1017). PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). The F8II.2210 peptide was purchased from AnaSpec (34801 Campus Drive, Fremont, CA 94555). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride.
Solution 2: 10 mg / mL F8II.2210 (TASSYFTNMFATWSPSKAR) and 100 mg / mL sucrose in 50 mM pH 2 phosphate buffer. This solution was prepared by dissolving sucrose in 50 mM pH 2 phosphate buffer and then adding the F8II.2210 peptide as a dry powder.
Solution 3: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.
Solution 4: 70 mM pH 8 phosphate buffer.

First, solution 1 (1.0 mL) and solution 2 (0.2 mL) are mixed in a small glass pressure tube and sonicated at 50% amplitude for 40 seconds using a Branson Digital Sonifier 250 to primary (W1). / O) An emulsion was prepared.
Solution 3 (3.0 mL) was then added to the primary emulsion and vortexed to make a crude dispersion, which was then sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. A W1 / O / W2) emulsion was formed.

The secondary emulsion was added to an open 50 mL beaker containing Solution 4 (30 mL) and stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in the suspension. The nanocarrier suspension was then transferred to a centrifuge tube and rotated at 75,600 rcf for 35 minutes to remove the supernatant and resuspend the pellet in phosphate buffered saline to allow the suspended nanocarriers. A part was washed. The washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The amount of F8II.2210 in the nanocarrier was determined using a fluorescamine-based assay. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Material Lactide: Glycolide ratio 1: 1 and intrinsic viscosity 0.24 dL / g PLGA was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027). (Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe in methylene chloride. This solution was prepared by dissolving PLGA and PLA-PEG-OMe in pure methylene chloride.
Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.
Solution 3: 70 mM phosphate buffer, pH 8.

Solution 1 (1.0 mL) and Solution 2 (3.0 mL) are mixed in a small glass pressure tube and sonicated at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250 to make an oil-in-water emulsion. did. The emulsion was added to an open 50 mL beaker containing Solution 3 (30 mL) and stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in the suspension. The nanocarrier suspension was then transferred to a centrifuge tube, rotated at 75,600 rcf for 40 minutes, the supernatant was removed and the pellet was resuspended in phosphate buffered saline to allow the suspended nanocarriers. A part was washed. The washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

The size of the nanocarrier was determined by dynamic light scattering. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Example 4: Evaluation of immune response to FVIII Synthetic nanocarriers (nanocarriers) attached to hemocoagulant factor VIII (FVIII) and rapamycin in hemophilia A mice (El6 mice) (n = 8 on day 0). Receive weekly combined intravenous (iv) injections of ID4) or empty nanoparticles (NP) (nanocarrier ID8) and FVIII or IVIG (intravenous immunoglobulin) and FVIII for 5 consecutive weeks I let you. A chromogenic FVIII activity assay kit (Coatest SP4 FVIII) was used to assess antibody recall response to FVIII one month after the final dose of nanocarriers attached to FVIII and rapamycin, as presented in FIG. The inhibitor titer was determined by the Bethesda assay.

To further assess the sustained tolerability induced by treatment of nanocarriers attached to FVIII and rapamycin, hemophilia A mice (El6 mice) (n = 8, 0 days), outlined in FIG. 6A. Synthetic nanocarriers and FVIII peptides (nanocarriers IDs 5, 6 and 7 were mixed and injected together in a 1: 1: 1 peptide mass ratio) or nanocarriers attached to rapamycin (nanocarriers) as presented. ID4) and FVIII protein or empty NP (nanocarrier ID8) and FVIII or IVIG (intravenous immunoglobulin) and FVIII in combination i. v. Injections were given weekly for 5 weeks. On days 57, 81 and 125, mice were challenged with FVIII (iv or ip) in the absence of further treatment. Anti-FVIII antibody levels were determined by ELISA. The results at the selected time points are presented in FIG. 6B. The data at each time point is expressed as mean ± SEM. For statistical analysis, student's t-test with bilateral distribution was used to compare the nanocarrier group (using proteins or peptides) with the empty nanoparticle control group. Between the nanocarrier ID4 having a protein and the empty NP group, * p <0.05 and ** p <0.01, the nanocarriers and peptides (nanocarrier IDs 5, 6 and 7) and the empty NP group Between # p <0.05 and ## p <0.01. All injections of FVIII were 1 μg per injection. All injections of nanocarrier ID4 were 100 μg per injection.

The data show that administration of an immunosuppressant attached to a synthetic nanocarrier with FVIII or a peptide thereof can reduce the anti-Factor VIII antibody response. This was true even when immunosuppressant synthetic nanocarriers were used in combination with non-adherent factor VIII proteins as well as factor VIII peptides attached to synthetic nanocarriers. The data also show that such administration may result in improved activity of Factor VIII. This demonstrates that the combined administration of immunosuppressants attached to synthetic nanocarriers reduces the unwanted immune response to the protein and improves efficacy. Finally, these results support the establishment of the protocol provided herein.

Example 5: Evaluation of response to HUMIRA / adalimumab Control C57BL / 6 same-age (5-6 weeks) females with 60 μg HUMIRA once weekly until day 29 (d0, 7, 14, 22 across all groups and conditions) , 29) Subcutaneous (sc) injection in the forefoot. Another group received similar injections, but 0.9 mg of rapamycin-attached nanocarriers (nanocarrier IDs 1, 2 or 3) were mixed with the solution of HUMIRA on day 0 of antigen stimulation. The results presented in FIG. 7A show antibody titers in the blood of all animals on day 21. Control animals exhibit a strong antibody response to HUMIRA, but treated animals remain completely negative even after 20 days and two untreated injections of HUMIRA. Animals received another challenge on one hind limb and saline on the other hind limb on day 29 to test a topical antibody-mediated type I hypersensitivity response. To this end, hindlimb thickness was measured with the help of calipers 1 hour after injection. The difference in the thickness of the two limbs is shown in FIG. 7B. Similar to antibody results, treatment with nanocarriers eliminated the inflammatory response induced by topical administration of HUMIRA.

These results can be achieved by administering an immunosuppressant attached to a synthetic nanocarrier with HUMIRA to reduce unwanted anti-HUMIRA antibody responses, as well as by administering HUMIRA without an immunosuppressant nanocarrier composition. It has been shown that the unwanted inflammatory response obtained can be ruled out. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 6: Evaluation of response to keyhole limpet hemocyanin (KLH) Control C57BL / 6 same-age (5-6 weeks) females with 200 μg of keyhole limpet hemocyanin (KLH) on day 0 or 21. Intravenous (iv) injection was performed in the tail vein once weekly until the 34th (d0, 7, 14, 21, 28, 34). Another group received similar injections from days 0 to 34, but on days 0, 14 and 21 nanocarriers attached to 0.47 mg rapamycin (nanocarrier IDs 1, 2 or 3). Was mixed with a solution of KLH, after which injections of KLH alone were received weekly (d21, 28, 34) for days 21-34. The results presented in FIG. 8 show antibody titers in the blood of all animals at the time of display. _{Control animals that initiated immunization on day 0 or 21 had a very similar response (EC 50} = 3-5) after 3 injections (26th and 40th days, respectively) using the same amount of KLH. × 10 ³ ) is shown. In contrast, animals that received three injections of nanocarriers remain completely negative after three injections of KLH alone.

The data indicate that administration of an immunosuppressant attached to a synthetic nanocarrier with KLH may reduce the anti-KLH antibody response, and that reduction may continue after administration of KLH after nanocarrier treatment. It shows that. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 7: Evaluation of response to ovalbumin C57BL / 6 same-age (5-6 weeks) females at -21 and -13 days with 1.2 mg non-adherent nanoparticles (NP [empty]) (nanocarrier ID 8) , Free rapamycin (frapa) (100 μg), 22 μg free chicken ovalbumin (fOVA), frapa in combination with fOVA, 1.2 mg rapamycin attached nanocarrier alone or in combination with 22 μg fOVA. (NP [rapa], 100 μg rapamycin content) (nanocarrier ID 1, 2 or 3) in the tail vein i. v. I injected it. On day 0, all animals were given 2.5 μg of particulate OVA (pOVA) mixed with 2 μg of CpG in the hind limbs. c. Injection was followed by injection of 2.5 μg of pOVA on days 7 and 14. In the absence of either treatment (NP [empty]), animals exhibit a strong immune response to OVA that can be measured by anti-OVA IgG antibody titers. The antibody titer on day 22 shown in FIG. 9 is that two doses of synthetic nanocarrier (fOVA + NP [rapa]) co-administered with OVA in the same solution, one injection of OVA + CpG and OVA alone. It has been demonstrated that it was effective in preventing antibody formation against OVA even after two injections.

The data show that administration of an immunosuppressant attached to a synthetic nanocarrier with an OVA can reduce the anti-OVA antibody response, and that reduction continues even after administration of the OVA in combination with a strong adjuvant. Shows that you will get. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 8: Evaluation of response to KRYSTEX XA On days 21 and -14, C57BL / 6 same-age (5-6 weeks) female controls were subjected to PBS in the tail vein i. v. Injections were made, while the treatment group was injected with 0.9 mg of rapamycin-attached nanocarriers (nanocarrier IDs 1, 2 or 3) in combination with 40 μg of KRYSTEX XA. All animals were s. Of 100 μg KRYSTEX XA combined with 20 μg CpG weekly from day 0 to day 28. c. The injection was given to the hind limbs (d0, 7, 12, 28). Control animals exhibit an immune response against KRYSTEX XA that can be measured by anti-KRYSTEX XAIgM antibody titers. The results in FIG. 10 show that treatment of animals with synthetic nanocarriers co-administered with KRYSTEX XA in the same solution was effective in preventing antibody formation to KRYSTEX XA over time. Treated animals did not show an anti-KRYSTEXXA response even after 5 injections of KRYSTEXXA + CpG without nanocarriers.

The data show that administration of an immunosuppressant attached to a synthetic nanocarrier with KRYSTEXXA can reduce the anti-KRYSTEXXA antibody response, and that such reduction is even after administration of KRYSTEXXA with a strong adjuvant after nanocarrier treatment. , Indicates that it can continue as it is. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 9: Evaluation of response to ovalbumin and KLH C57BL / 6 same-age (5-6 weeks) female control group once weekly for 49 days (d0,7,14,20,28,35,42,49) , 2.5 μg of particulate ovalbumin (pOVA) in immunogenic form in the tail vein i. v. Inject 2 μg of keyhole limpet hemocyanin (KLH) in the hind limbs. c. I injected it. Other groups of animals received pOVA and KLH on days 0, 7 and 14 with nanocarriers (nanocarrier IDs 1, 2 or 3) attached to 0.2 mg rapamycin in the same pathway and amount. It was mixed and received, followed by injection of pOVA between days 20-42 (same amount as before). Control animals exhibit a strong immune response against OVA and KLH that can be measured by anti-OVA or anti-KLHIgG antibody titers. The antibody titer on day 54 shown in FIG. 11 is effective in that three doses of synthetic nanocarriers co-administered with pOVA in the same solution reduce and prevent antibody formation to OVA over time. It was, but proved to be ineffective against KLH (injected cc elsewhere). Treated animals did not show an anti-OVA response after 5 injections of pOVA alone.

The data show that co-administration of immunosuppressive agents attached to synthetic nanocarriers with proteins can reduce anti-protein antibody responses, but such responses are specific for proteins administered by the same route. ing. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 10: Evaluation of response to KLH Control C57BL / 6 same-age (5-6 weeks) females were given 200 μg of keyhole limpet hemocyanin (KLH) once weekly for 63 days (d0, 7, 14, 21, 28). , 35, 42, 49, 56, 63), i. v. I injected it. Other groups received similar injections, but on days 0, 7, 14 and 21, 0.9 mg of rapamycin-attached nanocarriers (nanocarrier IDs 1, 2 or 3) were mixed with a solution of KLH. Subsequently, KLH was injected 6 times (equal dose) between the 28th and 63rd days. Control animals exhibited an injection-induced anaphylactic response with a strong response to KLH that could be measured by anti-KLHIgG antibody titers. The results in FIG. 12A show antibody titers in the blood of all animals at the time of display and present the anaphylaxis scores induced by injection of the antigen in FIG. 12B. Four doses of synthetic nanocarriers co-administered with KLH were effective in the long-term reduction and prevention of antibody formation and anaphylaxis. In fact, treated animals did not exhibit an anti-KLH response even after four injections of KLH alone (Day 26), which was generally sufficient to generate a response in control animals.

These results indicate that the compositions provided herein can reduce or prevent antibody formation and anaphylaxis over a long period of time when co-administered with a protein over a period of time. Anaphylaxis scores were determined by 3 independent observers as follows: 0 = no sign, l = lethargy, 2 = lethargy and inability to right, 3 = moribund.

These results result from administration of immunosuppressive agents attached to synthetic nanocarriers with KLH, which can reduce unwanted anti-KLH antibody responses and result from administration of KLH without immunosuppressive nanocarrier compositions. It shows that the unwanted anaphylactic reactions that can occur can be ruled out. This demonstrates the reduction of unwanted immune responses to proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. Finally, these results support the establishment of the protocol provided herein.

Example 11: Evaluation of anti-HUMIRA immune response in arthritic animals Transgenic animals that overexpress the human tumor necrosis factor alpha (huTNFαTg) develop progressive rheumatoid arthritis within 20 weeks of birth. This process can be prevented by using the fully human anti-human TNFα antibody HUMIRA / adalimumab. However, repeated doses of the initial therapeutic dose of HUMIRA (60 μg) lead to anti-drug antibody formation (ADA) that neutralizes the therapeutic effect. And only very high doses can maintain the therapeutic effect and overcome the neutralization of the immune response. For example, in the example of such a mouse model, it is believed that about 200 μg is required to achieve maximum inhibition of inflammation (Binder et al., Arthritis & Rheumatism, Vol. 65 (No. 3), March 2013, pp. 608-617).

First huTNFαTg 5-week-old female animals with saline (PBS) or 60 μg of HUMIRA weekly or a mixture of HUMIRA and 0.87 mg of rapamycin-attached nanocarriers (nanocarrier ID1, 2 or 3). 7 injections (0, 7, 14, 21, 28, 35, 42 days, 5-12 weeks old) followed by 3 injections of HUMIRA alone (equal dose) (49-63 days, 13) ~ 15 weeks of age) in the subscapular region. c. I injected it. The results in FIG. 13A show antibody titers in the blood of all animals at day 21. Control animals that did not receive HUMIRA did not have anti-HUMIRA titers as expected, whereas strong antibody responses could be observed in controls that received only HUMIRA. In contrast, animals treated with nanocarriers remain completely negative after three injections of untreated HUMIRA.

Monitoring of the limbs of these animals revealed a progressive disease that was already apparent at 10 weeks of age in control animals. Animals treated with HUMIRA alone had a significant blockage of disease progression, but the addition of nanocarriers to this regimen dramatically blocked the appearance of signs of arthritis. The scores here are 1) representing synovitis, joint exudate and soft tissue swelling, 2) including proliferation of inflamed synovial tissue that grows into the joint cavity and destroys cartilage, and 3) extensive cartilage. Independent of those exhibiting loss of cartilage, erosion and deformation around the periphery of the joint, 4) almost final stage of disease with joint fiber or bone stiffness that terminates its functional lifespan. Shows the total of 4 graders.

These results indicate that administration of immunosuppressants attached to synthetic nanocarriers with HUMIRA can reduce the unwanted anti-HUMIRA antibody response. The effect of the combination dose is also demonstrated by a further reduced arthritis score demonstrating the improved efficacy of HUMIRA. This demonstrates not only the reduction of unwanted immune responses to therapeutic proteins, but also the improved efficacy of the combined administration of immunosuppressants attached to synthetic nanocarriers. This also demonstrates that higher doses of therapeutic agents are not required for the concomitant administration of the invention provided herein.

In addition, based on the level of reduction in arthritis score with the same dose of HUMIRA, it is improved compared to the amount of HUMIRA that would be required without the combined administration of the immunosuppressive drug doses provided herein. A reduced amount of HUMIRA that will provide good efficacy can also be used. The amount of HUMIRA used in this example is 60 μg, which is about 200 μg used in the art to achieve maximum inhibition of inflammation by HUMIRA (Binder et al., Arthritis & Rheumatism, Vol. 65 (No. It is also important to note that this was significantly reduced compared to 3), March 2013, pp. 608-617). Finally, these results support the establishment of the protocol provided herein.

Example 12: Mesoporous silica nanoparticles with ibuprofen attached (prophetic)
Mesoporous SiO ₂ nanoparticle cores are made through a sol-gel process. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g) is dissolved in deionized water (500 mL), then 2 M aqueous NaOH solution (3.5 mL) is added to the CTAB solution. The solution is stirred for 30 min and then tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. The resulting gel is stirred at a temperature of 80 ° C. for 3 hours. The white precipitate to form is captured by filtration, then washed with deionized water and dried at room temperature. The remaining surfactant is extracted from the particles by suspending overnight in an ethanolic solution of HCl. The particles are washed with ethanol, centrifuged and redispersed under ultrasound. This cleaning procedure is repeated two additional times.

SiO ₂ nanoparticles are then functionalized with an amino group using (3-aminopropyl) -triethoxysilane (APTMS). To do this, the particles are suspended in ethanol (30 mL) and APTMS (50 μl) is added to the suspension. After leaving the suspension at room temperature for 2 hours, ethanol is added periodically to bring the suspension to a boil for 4 hours while keeping the volume constant. The residual reactants are removed by 5 cycles of washing by centrifugation and redispersion in pure ethanol.

As a separate reaction, gold seeds with a diameter of 1 to 4 nm are prepared. First all the water used in this reaction is deionized and then distilled from the glass. Add water (45.5 mL) to a 100 mL round bottom flask. With stirring, 0.2 M aqueous NaOH (1.5 mL) followed by 1% aqueous tetrakis (hydroxymethyl) phosphonium chloride (THPC) solution (1.0 mL) is added. Two minutes after adding the THPC solution, add at least 15 min aged 10 mg / mL chloroauric acid aqueous solution (2 mL). Purify the gold seeds through dialysis against water.

To form the core-shell _{nanocarriers, the amino-functionalized SiO 2} nanoparticles formed above are first mixed with gold seeds for 2 hours at room temperature. Gold-decorated SiO ₂ particles are collected through centrifugation and mixed with an aqueous solution of chloroauric acid and potassium bicarbonate to form a gold shell. The particles are then washed by centrifugation and redispersion in water. Ibuprofen is added by suspending the particles in a solution of sodium ibuprofen (1 mg / L) for 72 hours. Free ibuprofen is then washed from the particles by centrifugation and redispersion in water.

Example 13: Liposomes containing cyclosporin A (prophetic)
Liposomes are formed using thin film hydration. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol), cholesterol (32 μmol) and cyclosporin A (6.4 μmol) are dissolved in pure chloroform (3 mL). This lipid solution is added to a 50 mL round bottom flask and the solvent is evaporated on a rotary evaporator at a temperature of 60 ° C. The flask is then flushed with nitrogen gas to remove any residual solvent. Phosphate buffered saline (2 mL) and 5 glass beads are added to the flask and the lipid membrane is hydrated by shaking at 60 ° C. for 1 h to form a suspension. The suspension is transferred to a small pressure tube and sonicated at 60 ° C. for 4 cycles of 30 s pulses with a delay of 30 seconds between each pulse. The suspension is then allowed to stand at room temperature for 2 hours to completely hydrate. The liposomes are washed by centrifugation followed by resuspension in fresh phosphate buffered saline.

Example 14: Preparation of gold nanocarrier (AuNC) containing rapamycin (prophetic)
Preparation of HS-PEG-rapamycin:
A dry DMF solution of disulfide PEGylate (1.0 eq), rapamycin (2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) is stirred overnight at rt. The insoluble dicyclohexylurea is removed by filtration, the filtrate is added to isopropyl alcohol (IPA) to precipitate the PEG-disruifido-di-rapamycin ester, washed with IPA and dried. The polymer is then treated with tris (2-carboxyethyl) phosphine hydrochloride in DMF to reduce the PEG disulfide to a thiol PEG rapamycin ester (HS-PEG-rapamycin). The resulting polymer is recovered by precipitation from IPA as described above, dried and analyzed by 1 H-NMR and GPC.

Formation of gold NC (AuNC):
500 mL of an aqueous solution of 1 mM HAuCl ₄ is heated under reflux for 10 minutes with vigorous stirring in a 1 L round bottom flask with a condenser. Then 50 mL of a 40 mM trisodium citrate solution is added rapidly to the stirred solution. The resulting concentrated wine red solution is kept at reflux for 25-30 min to remove heating and allow the solution to cool to room temperature. The solution is then filtered through a 0.8 μm membrane filter to obtain an AuNC solution. AuNC is characterized using visible spectroscopy and transmission electron microscopy. AuNC is capped by citrate at a diameter of about 20 nm and has a peak absorption at 520 nm.

AuNC complex with HS-PEG-rapamycin:
150 μl of a solution of HS-PEG-rapamycin (10 μΜ in 10 mM pH 9.0 carbonate buffer) was added to 1 mL of 20 nm diameter citrate-capped gold nanocarrier (1.16 nM) and a 2500: 1 thiol: gold molar ratio. To generate. The mixture is stirred under argon at room temperature for 1 hour to completely replace the thiol with the citrate on the gold nanocarrier. AuNC with PEG-rapamycin on the surface is then purified by centrifugation at 12,000 g for 30 minutes. After tilting the supernatant, the pellet containing AuNC-S-PEG-rapamycin is washed with 1 x PBS buffer. The purified gold-PEG-rapamycin nanocarrier is then resuspended in a suitable buffer for further analysis and bioassay.

Example 15: Liposomes containing rapamycin and ovalbumin (prophetic)
Liposomes are formed by thin film hydration. l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol), cholesterol (32 μmol) and rapamycin (6.4 μmol) are dissolved in pure chloroform (3 mL). This lipid solution was added to a 10 mL glass tube, the solvent was removed under a stream of nitrogen gas, and the mixture was dried under vacuum for 6 hr. Multilayer vesicles are obtained by hydration of the membrane with 2.0 mL of 25 mM MOPS buffer pH 8.5 containing an excess of ovalbumin. Vortex the tube until the lipid membrane is detached from the tube surface. A 10 cycle of freezing (liquid nitrogen) and thawing (30 ° C. water bath) is applied to break the multilayer vesicles into a single layer. The sample is then diluted to 1 mL in 25 mM MOPS buffer pH 8.5. The resulting liposome size is homogenized by extrusion by passing the sample through a 200 nm pore polycarbonate filter 10 times. The resulting liposomes are then used for further analysis and bioassay.

Example 16: Tolerant response to HUMIRA prevents the formation of a neutralizing anti-HUMIRA response in arthritic animals. The material rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (185 Wilson Street, Framingham, MA 01702). Product code R1017). PLGA with a lactide: glycolide ratio of 3: 1 and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027) ( Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride. Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

Nanocarriers were prepared using oil-in-water emulsions. Prepare an O / W emulsion by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. did. The O / W emulsion was added to a beaker containing a 70 mM pH 8 phosphate buffer solution and stirred at room temperature for 2 hours to evaporate methylene chloride to form nanocarriers. The nanocarrier suspension was transferred to a centrifuge tube and centrifuged at 75,600 xg and 4 ° C. for 50 min, the supernatant was removed, and the pellet was resuspended in phosphate buffered saline to resuspend the nanocarrier. Was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final nanocarrier dispersion of about 10 mg / mL.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Transgenic animals that overexpress the human tumor necrosis factor alpha (huTNFαTg) develop progressive rheumatoid arthritis within 20 weeks of birth. This process can be prevented by using the fully human anti-human TNFα antibody adalimumab or HUMIRA. However, repeated doses of the initial therapeutic dose of HUMIRA (60 μg) lead to anti-drug antibody formation (ADA) that neutralizes the therapeutic effect.

Same-age HuTNFαTg 5-week-old female animals are injected with saline (PBS) or 60 μg of HUMIRA weekly or the first 7 injections of a mixture of HUMIRA and 0.87 mg tolerant nanoparticles (Days 0-42, 5). ~ 11 weeks of age) followed by 10 weekly injections of HUMIRA alone (equal dose) (49th to 107th days, 12 to 20 weeks of age) in the subscapular region. c. I injected it. The protocol is shown in FIG.

The results in FIG. 15 (left panel) show antibody titers in the blood of all animals at different time points. Control sham-treated animals do not have titers as expected, while strong anti-HUMIRA antibody responses can be observed in animals that receive only HUMIRA. Treatment with tolerant nanocarriers from 5 to 11 weeks of age exhibits anti-HUMIRA titers even after 10 injections (12th to 20th week) of HUMIRA without tolerant treatment. It led to a complete resistance to. Monitoring of the limbs of these animals revealed a progressive disease that was already apparent at 10 weeks of age. Although HUMIRA alone had a significant blockade of disease progression, the addition of tolerant nanocarriers to this regimen dramatically blocked the appearance of signs of arthritis. The scores here are 1) representing synovitis, joint exudate and soft tissue swelling, 2) including proliferation of inflamed synovial tissue that grows into the joint cavity and destroys cartilage, and 3) extensive cartilage. Independent of those exhibiting loss of cartilage, erosion and deformation around the periphery of the joint, 4) almost final stage of disease with joint fiber or bone stiffness that terminates its functional lifespan. Shows the average total of 4 graders.

These results indicate that the compositions provided herein can reduce or prevent antibody formation to biotherapeutic agents when co-administered with a protein over a period of time and therefore their efficacy over the long term. And show that it improves the treatment window. These results also show that administration of immunosuppressants attached to synthetic nanocarriers with HUMIRA can reduce unwanted anti-HUMIRA antibody responses. The effect of the combination dose is also demonstrated by a further reduced arthritis score demonstrating the improved efficacy of HUMIRA. This also demonstrates the improved efficacy as well as the reduction of unwanted immune responses to therapeutic proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers.

This also demonstrates that higher doses of therapeutic agents are not required for the concomitant administration of the invention provided herein. In addition, improvements compared to the amount of HUMIRA that would be required without the combined administration of immunosuppressive drug doses provided herein, based on the level of reduction in arthritis score with the same dose of HUMIRA A reduced amount of HUMIRA that will provide the desired efficacy can also be used. The amount of HUMIRA used in this example is 60 μg, which is about 200 μg used in the art to achieve maximum inhibition of inflammation by HUMIRA (Binder et al., Arthritis & Rheumatism, Vol. 65 (No. It is also important to note that this was significantly reduced compared to 3), March 2013, pp. 608-617). Finally, these results support the establishment of the protocol provided herein.

Example 17: Evaluation of anti-HUMIRA immune response Material rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (product code # R1017). PLGA with a lactide: glycolide ratio of 3: 1 and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211) (product code 7525 DLG 7A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.5 DL / g were purchased from Lakeshore Biochemicals (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027) ( Product code 1.41350).

Method The solution was prepared as follows:
Solution 1: 75 mg / mL PLGA, 25 mg / mL PLA-PEG-OMe and 12.5 mg / mL rapamycin in methylene chloride. This solution was prepared by dissolving PLGA, PLA-PEG-OMe and rapamycin in pure methylene chloride. Solution 2: Polyvinyl alcohol, 50 mg / mL in 100 mM pH 8 phosphate buffer.

Nanocarriers were prepared using oil-in-water emulsions. Prepare an O / W emulsion by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small pressure tube and sonicating at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. did. The O / W emulsion was added to a beaker containing a 70 mM pH 8 phosphate buffer solution and stirred at room temperature for 2 hours to evaporate methylene chloride to form nanocarriers. The nanocarrier suspension was transferred to a centrifuge tube and centrifuged at 75,600 xg and 4 ° C. for 50 min, the supernatant was removed, and the pellet was resuspended in phosphate buffered saline to resuspend the nanocarrier. Was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline to give a final nanocarrier dispersion of about 10 mg / mL.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

Rapamycin-containing nanocarriers were generated using the materials and methods described above. The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

The anti-HUMIRA immunity and neutralization response in arthritic animals was evaluated. Transgenic animals that overexpress the human tumor necrosis factor alpha (huTNFαTg) develop progressive rheumatoid arthritis within 20 weeks of birth. This process can be prevented by using the fully human anti-human TNFα antibody adalimumab or HUMIRA. However, repeated doses of the initial therapeutic dose of HUMIRA (60 μg) lead to anti-drug antibody formation (ADA) that neutralizes the therapeutic effect. A larger dose of Humira (200 μg) may be injected to overcome this antagonistic immune response and enable the therapeutic effect of Humira.

Same-age HuTNFαTg 5-week-old female animals with saline (PBS) or 60 μg or 200 μg of HUMIRA weekly (weeks 5-10) or a mixture of 60 μg Humila and 0.87 mg tolerant nanoparticles. Either the first 3 injections (5-7 weeks of age) followed by 3 weekly injections of HUMIRA alone (equal dose) (8-10 weeks of age) were given to the subscapular region. c. I injected it. The results in FIG. 17 (left panel) show antibody titers in the blood of all animals at different time points. Control sham-treated animals do not have titers as expected, while strong anti-HUMIRA antibody responses can be observed in animals that receive only HUMIRA. Treatment with 5-7 week old nanocarriers led to complete resistance to manifesting anti-HUMIRA titers, even after 3 injections of untreated HUMIRA (8th-10 weeks). Notably, three injections of HUMIRA led to very high titers in control animals (week 7 titers), but three similar injections in animals treated with nanocarriers (7th week titer). (6 times in total) were totally reluctant to show titers (week 10). FIG. 16 illustrates a dosing regimen.

Monitoring of the limbs of these animals revealed a progressive disease that was already apparent at 6 weeks of age. HUMIRA alone had a significant blockade of disease progression, but the addition of nanocarriers to this regimen dramatically blocked the appearance of signs of arthritis. Scores (FIG. 17 (right panel)) 1) represent synovitis, joint exudate and soft tissue swelling, 2) include proliferation of inflamed synovial tissue that grows into the joint cavity and destroys cartilage, 3) Extensive loss of cartilage, erosion and deformation around the periphery of the joint, 4) almost final stage of disease with joint fiber or bone stiffness that terminates its functional life. Represents the average sum of four independent graders.

These results indicate that the methods and compositions provided herein can reduce or prevent the formation of antibodies to therapeutic proteins and improve their effectiveness. Specifically, as described above, these results indicate that administration of an immunosuppressant attached to a synthetic nanocarrier with HUMIRA can reduce the unwanted anti-HUMIRA antibody response. The effect of the combination dose is also demonstrated by a further reduced arthritis score demonstrating the improved efficacy of HUMIRA. This also demonstrates the improved efficacy as well as the reduction of unwanted immune responses to therapeutic proteins by the combined administration of immunosuppressants attached to synthetic nanocarriers. This also demonstrates that the concomitant administration of the invention provided herein does not require higher doses of the therapeutic agent.

In addition, an improvement compared to the amount of HUMIRA that would be required without the combined administration of the immunosuppressive drug doses provided herein, based on the level of reduction in arthritis score with the same dose of HUMIRA. A reduced amount of HUMIRA that would provide the desired efficacy could also be used. The amount of HUMIRA used in this example is 60 μg, which is about 200 μg used in the art to achieve maximum inhibition of inflammation by HUMIRA (Binder et al., Arthritis & Rheumatism, Vol. 65 (No. It is also important to note that this was significantly reduced compared to 3), March 2013, pp. 608-617). Finally, these results support the establishment of the protocol provided herein.

Example 18: Antigen-specific tolerant response to chicken ovalbumin with encapsulated rapamycin NP [rapa] material and method material rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702) (product code # R1017). .. PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.50 DL / g were purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) ( Product code 100DL mPEG 5000 5CE). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa · s) was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027) ( Product code 1.41350). Cellgro Phosphate-buffered saline 1X (PBS1x) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

Method The solution was prepared as follows:
A polymer and rapamycin mixture was prepared by dissolving 75 mg PLGA per mL of solution 1: 25 mg PLA-PEG-Ome per mL and 12.5 mg rapamycin per mL in dichloromethane. Solution 2: Polyvinyl alcohol was prepared at 50 mg / mL in 100 mM pH 8 phosphate buffer.

O / W emulsions are formed by combining solution 1 (1.0 mL) and solution 2 (3.0 mL) in a small glass pressure tube and sonicating at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. Prepared. This O / W emulsion was added to an open beaker containing 70 mM pH 8 phosphate buffer (60 mL). Three additional identical O / W emulsions were prepared and added to the same beaker as the first. These were then stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers. The nanocarrier suspension is then transferred to a centrifuge tube and centrifuged at 75,600 xg and 4 ° C. for 35 minutes, the supernatant is removed and the pellet is resuspended in PBS 1x to allow the nanocarrier. Was washed. The washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The same formulation was prepared as above in a separate beaker and combined with the first after the wash step. The mixed nanocarrier solution was then filtered using Pall's 1.2 μm PES membrane syringe filter (part number 4656)) and stored at −20 ° C.

The size of the nanocarrier was determined by dynamic light scattering. The amount of rapamycin in the nanocarrier was determined by HPLC analysis. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

NP [OVA] Material and Method Material Ovalbumin protein was purchased from Worthington Biochemical Corporation (730 Vassar Avenue, Lakewood, NJ 08701) (product code LS003054). PLGA with 54% lactide and 46% glycolide content and intrinsic viscosity of 0.24 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an intrinsic viscosity of 28,000 Da Mw, 0.38 dL / g were purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 4CE). EMPROVE® Polyvinyl Alcohol 4-88, USP, 85-89% hydrolyzed, viscosity 3.4-4.6 mPa. s, was purchased from EMD Chemicals Inc. (480 South Democrat Road Gibbstown, NJ 08027) (product code 1.41350.1001). Cellgro Phosphate-buffered saline 1X (PBS1x) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

Method The solution was prepared as follows:
Solution 1: 50 mg / mL ovalbumin protein was prepared in 10 mM phosphate buffer pH 8 with 10 wt% sucrose. Solution 2: PLGA was prepared by dissolving 100 mg of PLGA per mL of dichloromethane in a fume hood. Solution 3: PLA-PEG-OMe was prepared by dissolving 100 mg of PLA-PEG-OMe per mL of dichloromethane in a fume hood. Solution 4: Polyvinyl alcohol, 100 mM phosphate buffer 65 mg / mL in pH 8.

First, a primary (W1 / O) emulsion was prepared by mixing solutions 1 to 3. Combine solution 1 (0.2 mL), solution 2 (0.75 mL) and solution 3 (0.25 mL) in an ice water bath> 4 minutes precooled in a small glass pressure tube and use the Branson Digital Sonifier 250. Sonication was performed on an ice bath at 50% amplitude for 40 seconds. Solution 4 (3 mL) was then added to the primary emulsion and vortex mixed to make a milky dispersion, then sonicated on an ice bath at 30% amplitude for 60 seconds using a Branson Digital Sonifier 250. A secondary (W1 / O / W2) emulsion was formed. This secondary emulsion was added to an open 50 mL beaker containing PBS 1x (30 mL). A second identical double emulsion formulation was prepared as described above and added to the same 50 mL beaker as the first. The two preparations were stirred at room temperature for 2 hours to evaporate dichloromethane to form nanocarriers in suspension. A portion of the suspended nanocarrier was washed by transferring the nanocarrier suspension to a centrifuge tube, rotating at 75,600 rcf for 50 minutes, removing the supernatant and resuspending the pellet in PBS 1x. This washing procedure was repeated and then the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The suspension was frozen and stored at −20 ° C. until use.

NP [GSK1059615] Materials and Method Materials
GSK1059615 was purchased from MedChem Express (11 Deer Park Drive, Suite 102D Monmouth Junction, NJ 08852) (product code HY-12036). PLGA with a lactide: glycolide ratio of 1: 1 and an intrinsic viscosity of 0.24 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 5050 DLG 2.5A). PLA-PEG-OMe block copolymers with a methyl ether-terminated PEG block of approximately 5,000 Da and an overall intrinsic viscosity of 0.26 DL / g were purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211). Product code 100 DL mPEG 5000 5K-E). Cellgro Phosphate-buffered saline 1X pH 7.4 (PBS1x) was purchased from Corning (9345 Discovery Blvd. Manassas, VA 20109) (product code 21-040-CV).

Method The solution was prepared as follows:
Solution 1: PLGA (125 mg) and PLA-PEG-OMe (125 mg) were dissolved in 10 mL of acetone. Solution 2: GSK1059615 was prepared at 10 mg in 1 mL of N-methyl-2-pyrrolidinone (NMP).

Solution 1 (4 mL) and Solution 2 (0.25 mL) were combined in a small glass pressure tube and the mixture was added dropwise to a 250 mL round bottom flask containing 20 mL of ultrapure water under stirring. Nanocarriers were prepared. The flask was attached to a rotary evaporation device and acetone was removed under reduced pressure. A portion of the nanocarrier was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 rcf and 4 ° C. for 50 minutes, removing the supernatant and resuspending the pellet in PBS 1x. did. The washing procedure was repeated and the pellet was resuspended in PBS 1x to achieve a nanocarrier suspension with a nominal concentration of 10 mg / mL on a polymer basis. The washed nanocarrier solution was then filtered using Pall's 1.2 μm PES membrane syringe filter (part number 4656). The same nanocarrier solution was prepared as described above and stored with the first after the filtration step. This homogeneous suspension was frozen and stored at −20 ° C.

The size of the nanocarrier was determined by dynamic light scattering. The amount of GSK1059615 in the nanocarrier was determined by UV absorption at 351 nm. The total dry nanocarrier mass per 1 mL of suspension was determined by specific gravity measurement.

C57BL / 6 same-age (5-6 weeks) female mice on days -21 and -14 with saline (untreated), 1.2 mg rapamycin-containing nanocarrier (NP [rapa]) or 8 mg GSK1059615-added nano 1.1 mg of total ovalbumin-supplemented nanocarrier (NP [OVA]) combined with any of the carriers (NP [GSK1059615]) was applied in the tail vein i. v. I injected it.

On day 0, all animals were subjected to 25 μg particulate OVA (pOVA) mixed with 2 μg CpG in the hind limbs. c. Injections were then made, followed by injections of only 25 μg pOVA on days 7 and 14. The antibody titer was measured on the 21st day. In the absence of either treatment, animals exhibited a strong immune response to OVA that could be measured by anti-OVA IgG antibody titers. The antibody titers on day 21 shown in FIG. 18 are two doses of synthetic tolerant nanocarriers co-administered with encapsulated OVA in the same solution (NP [OVA] + NP [rapa] or NP [GSK1059615]. ]) Demonstrate that even after one injection of OVA + CpG and two injections of OVA alone, it was effective in reducing antibody formation to OVA.

These results indicate that encapsulated immunosuppressants (such as rapamycin and GSK1059615), when delivered in combination with a protein, can prevent the formation of antibodies to the protein against multiple challenges and for extended periods of time. ing. This does not only demonstrate the reduction of unwanted immune responses to proteins by the combined administration of two different types of immunosuppressants. Finally, these results support the establishment of the protocol provided herein.

Example 19: The method of the invention using a reduced pharmacodynamically effective dose (prophetic)
We will recruit 3,200 human subjects with rheumatoid arthritis for a series of clinical trials. In the pilot dose range exploration study, 1,200 subjects are divided into four arms (three different dose levels of placebo and the synthetic nanocarrier of Example 3). Each subject of each of the four arms was used in combination with either placebo or a synthetic nanocarrier to HUMIRA 40 mgs. c. Receive 2 rounds. The synthetic nanocarrier dose that most reduces the average level of anti-HUMIRA antibody in an arm is declared as the "immunosuppressive agent dose" for that study.

In another pilot test, the recruited human subjects are divided into four test arms of 500 subjects each. Placebo, HUMIRA and the synthetic nanocarriers of Example 3 are co-administered according to the table below (excluding test arm 1), but the synthetic nanocarriers are administered at immunosuppressant doses.

The target pharmacodynamic effect (“PD effect”) is evaluated, in which case it is the average of ACR 20, 50 and 70 responses for the subject of each test arm. Focusing on the PD effect of the subject in test arm 1, the HUMIRA dose in test arm 1 is optionally defined as the pharmacodynamically effective dose of HUMIRA (“PD effective dose”).
The PD effect of test arms 2-4 is then evaluated and the lowest dose at which the PD effect does not differ significantly from the PD effect of test arm 1 is declared as the reduced pharmacodynamically effective dose of HUMIRA.

In the application of the information established during the pilot study, reduced pharmacodynamically effective doses of HUMIRA were given to subjects diagnosed with rheumatoid arthritis and at risk of developing antibodies to HUMIRA, immunosuppressive doses (synthesis of Example 3). Containing nanocarriers) and administer in combination. In a further embodiment, a pilot study to guide the combined administration of HUMIRA ™ and the synthetic nanocarrier of Example 3 to human subjects diagnosed with rheumatoid arthritis and having or expected to have antibodies to HUMIRA. Prepare a protocol that uses the information established in it. This protocol is then used to guide the combined administration of reduced pharmacodynamically effective doses of HUMIRA to the synthetic nanocarriers of Example 3 in human subjects.

Example 20: Method of the present invention demonstrating improved pharmacodynamic effects (prophetic)
3,700 human subjects with rheumatoid arthritis will be recruited for a series of clinical trials. In the pilot dose range exploration study, 1,200 subjects are divided into four arms (three different dose levels of placebo and the synthetic nanocarrier of Example 3). Each subject in each of the four arms, in combination with either placebo or synthetic nanocarriers, HUMIRA 40 mgs. c. Receive 2 rounds. The synthetic nanocarrier dose that most reduces the average level of anti-HUMIRA antibody in an arm is declared as the "immunosuppressive agent dose" for that study.

In another pilot study, the recruited human subjects are divided into three test arms: two active test arms, each with 1000 subjects, and one placebo arm with 500 subjects. Placebo, HUMIRA and the synthetic nanocarriers of Example 3 are co-administered according to the table below (excluding test arm 1), but the synthetic nanocarriers are administered at immunosuppressant doses.

The target pharmacodynamic effect (“PD effect”) is evaluated, in which case it is the average of ACR 20, 50 and 70 responses for the subject of each test arm. Pay attention to the PD effect of the target in the test arm 1 and compare it with the PD effect in the test arm 2. Note any improvement in the pharmacodynamic effect of HUMIRA when co-administered with the immunosuppressant dose in test arm 2 when compared to the PD effect observed in test arm 1.

In the application of the information established during the pilot study, subjects diagnosed with rheumatoid arthritis and at risk of developing antibodies to HUMIRA will receive a standard 40 mg dose of HUMIRA and an immunosuppressive drug dose (containing the synthetic nanocarrier of Example 3). ) And administer in combination. In a further embodiment, a pilot to guide the combined administration of HUMIRA and the synthetic nanocarrier of Example 3 to a human subject who has been diagnosed with rheumatoid arthritis and is known to have or is believed to have antibodies to HUMIRA. Prepare a protocol that uses the information established during the test. This protocol is then used to guide the combined administration of HUMIRA and the synthetic nanocarrier of Example 3 to human subjects. Any improved pharmacodynamic effect following concomitant dosing will be recorded using the approach disclosed herein.

Example 21: Method of the present invention demonstrating improved pharmacodynamic effects (prophetic)
3,200 human subjects with chemotherapy-related anemia will be recruited for a series of clinical trials. In the pilot dose range exploration study, modified mRNA (“m mRNA”) encoding erythropoietin is prepared according to US patent application 2013/0115272 (de Fougerolles et al.). Divide the 1,200 subjects into four arms (three different dose levels of placebo and the synthetic nanocarrier of Example 3). Each subject in each of the four arms receives a therapeutic dose of mmRNA in combination with either placebo or a synthetic nanocarrier. The synthetic nanocarrier dose that most reduces the average level of anti-m mRNA antibody in an arm is declared as the "immunosuppressive agent dose" for that study.

In another pilot test, the recruited human subjects are divided into four test arms of 500 subjects each. Placebo, mmRNA and the synthetic nanocarrier of Example 3 are co-administered according to the table below (excluding test arm 1), but the synthetic nanocarrier is administered at an immunosuppressive dose.

The target pharmacodynamic effect (“PD effect”) is assessed, in this case the mean value of the chemotherapy-induced anemia response for each test arm subject. Focusing on the PD effect of the subject in test arm 1, the mmRNA dose in test arm 1 is optionally defined as the pharmacodynamically effective dose of mmRNA (“PD effective dose”).

The PD effect of test arms 2-4 is then evaluated and the lowest dose at which the PD effect does not differ significantly from the PD effect of test arm 1 is declared as the reduced pharmacodynamically effective dose of mmRNA. In the application of the information established during the pilot study, immunosuppressive drug doses (eg, eg 3) of immunosuppressive doses were given to subjects diagnosed with chemotherapy-related anemia and at risk of developing antibodies to mmRNA. Containing synthetic nanocarriers).

In a further embodiment, to guide the combined administration of mmRNA and the synthetic nanocarrier of Example 3 to a human subject who has been diagnosed with chemotherapeutic anemia and is known to have or is believed to have an antibody to mmRNA. To prepare a protocol that uses the information established during the pilot test. This protocol is then used to guide the combined administration of reduced pharmacodynamically effective doses of mmRNA and the synthetic nanocarriers of Example 3 to human subjects.

Example 22: Methods for Maintaining the Effectiveness of Biological Drugs During the Period of Multiple Dosing Biological drugs often lose their activity over time after multiple doses. A common cause of loss of effectiveness is the formation of ADA. ADA can be used, for example, by 1) improving drug clearance so that the PK of the drug after multiple doses is substantially lower than the PK of the drug after a single dose, or 2) the activity of the drug. It can cause a loss of drug efficacy by neutralizing the drug, resulting in the biological activity of the drug after multiple doses being substantially lower than the biological activity after a single dose. It is desirable to maintain the activity of the biological drug during multiple doses.

Transgenic mice expressing human TNF-α spontaneously develop arthritis over a period of 5 to 20 weeks of age. Mice were treated weekly with HUMIRA (60 μg / injection) with or without synthetic nanocarriers from Example 16 for 5-11 weeks. Panel B (FIG. 19) shows serum levels of HUMIRA after the first dose (day 1). In mice treated with HUMIRA alone, after 6 doses of HUMIRA, HUMIRA blood levels approach baseline and remain low until week 20 (Panel C, FIG. 19, black square). In contrast, mice treated with HUMIRA and synthetic nanocarriers showed similar HUMIRA serum levels after the first dose (Panel C, FIG. 19, blue triangle), and synthetic nanocarrier treatment of HUMIRA after multiple doses. It indicates that it has made it possible to maintain effective blood levels.

The reduction in serum HUMIRA blood levels in mice treated with HUMIRA alone can be attributed to the manifestation of ADA. By week 11, mice treated with HUMIRA alone exhibited high titers of anti-drug antibodies (Panel A, FIG. 19, black square symbol). In contrast, mice treated with synthetic nanocarriers show little or no anti-HUMIRA antibody response (Panel A, FIG. 19, blue triangle). The effect of multiple doses on reducing HUMIRA serum levels is evident in the arthritic disease scores of Example 16 as described above (Panel D, FIG. 19). Mice treated with HUMIRA alone show suboptimal attenuation of arthritis scores from weeks 10 to 20 (Panel D, FIG. 19, compare black squares with black circles). In contrast, mice treated with synthetic nanocarriers show strong inhibition of arthritis (Panel C, FIG. 19, blue triangle).

These results indicate that maintaining drug levels by multiple doses is important for drug efficacy, and that immunosuppressants attached to synthetic nanocarriers block drug levels by multiple doses by inhibiting the ADA response. Is displayed to maintain. Therefore, these results demonstrate the effect of repeated administration of therapeutic macromolecules in combination with immunosuppressants as provided herein. The results further demonstrate the ability to determine the desired therapeutic effect and / or protocol capable of performing a reduced ADA response in the subject.

Example 23: Method of the present invention demonstrating improved pharmacodynamic effects (prophetic)
3,700 human subjects with rheumatoid arthritis will be recruited for a series of clinical trials. In a pilot dose range exploration study, 1,200 subjects will be divided into four arms (three different dose levels of placebo and nanocrystalline rapamycin). Each subject of each of the four arms was HUMIRA 40 mgs with either placebo or nanocrystalline rapamycin. c. Receive 2 rounds. The nanocrystalline rapamycin dose that most reduces the average level of anti-HUMIRA antibody in an arm is declared as the "immunosuppressive drug dose" for that study.

In another pilot study, the recruited human subjects are divided into three test arms: two active test arms, each with 1000 subjects, and one placebo arm with 500 subjects. Placebo, HUMIRA and nanocrystalline rapamycin are co-administered according to the table below (excluding test arm 1), but synthetic nanocarriers are administered at immunosuppressant doses.

The target pharmacodynamic effect (“PD effect”) is assessed, in this case the average of ACR 20, 50 and 70 responses for the subject of each test arm. Pay attention to the PD effect of the target in the test arm 1 and compare it with the PD effect in the test arm 2. Note any improvement in the pharmacodynamic effect of HUMIRA when co-administered with the immunosuppressant dose in test arm 2 when compared to the PD effect observed in test arm 1.

In the application of the information established during the pilot study, for subjects diagnosed with rheumatoid arthritis and at risk of developing antibodies to HUMIRA, a standard 40 mg dose of HUMIRA, an immunosuppressive drug dose (containing nanocrystalline rapamycin). Administer in combination with. In a further embodiment, a pilot study to guide the combined administration of HUMIRA and nanocrystalline rapamycin to human subjects who have been diagnosed with rheumatoid arthritis and are known or believed to have antibodies to HUMIRA. Prepare a protocol that uses the information established in it. This protocol is then used to guide the combined administration of HUMIRA and nanocrystalline rapamycin to human subjects. Any improved pharmacodynamic effect following concomitant dosing will be recorded using the approach disclosed herein.

Example 24: Method of the present invention demonstrating improved pharmacodynamic effects (prophetic)
3,200 human subjects with chemotherapy-related anemia will be recruited for a series of clinical trials. In the pilot dose range exploration study, modified mRNA (“m mRNA”) encoding erythropoietin is prepared according to US patent application 2013/0115272 (de Fougerolles et al.). Divide the 1,200 subjects into four arms (three different dose levels of placebo and nanocrystalline rapamycin). Each subject in each of the four arms receives a therapeutic dose of mmRNA in combination with either placebo or nanocrystalline rapamycin. The nanocrystalline rapamycin dose that most reduces the average level of anti-m mRNA antibody in an arm is declared as the "immunosuppressive agent dose" for that study.

In another pilot test, the recruited human subjects are divided into four test arms of 500 subjects each. Placebo, mmRNA and the synthetic nanocarrier of Example 3 are co-administered according to the table below (excluding test arm 1), while nanocrystalline rapamycin is administered at an immunosuppressant dose.

The target pharmacodynamic effect (“PD effect”) is assessed, in this case the mean value of the chemotherapy-induced anemia response for each test arm subject. Focusing on the PD effect of the subject in test arm 1, the mmRNA dose in test arm 1 is optionally defined as the pharmacodynamically effective dose of mmRNA (“PD effective dose”).

The PD effect of test arms 2-4 is then evaluated and the lowest dose at which the PD effect does not differ significantly from the PD effect of test arm 1 is declared as the reduced pharmacodynamically effective dose of mmRNA. In the application of the information established during the pilot study, immunosuppressive drug doses (nanocrystals) were given to subjects diagnosed with chemotherapy-related anemia and at risk of developing antibodies to mmRNA. Containing sex rapamycin).

In a further embodiment, to guide the combined administration of mmRNA and nanocrystalline rapamycin to human subjects who have been diagnosed with chemotherapeutic anemia and are known to have or are believed to have antibodies to mmRNA. Prepare a protocol that uses the information established during the pilot test. This protocol is then used to guide the combined administration of reduced pharmacodynamically effective doses of mmRNA and nanocrystalline rapamycin to human subjects.

Claims (69)

Providing an immunosuppressant dose, where the immunosuppressant dose is attached to a synthetic nanocarrier; and
A method comprising administering a reduced pharmacodynamically effective dose of a therapeutic polymer in combination with an immunosuppressive drug dose to a subject expected to have an anti-therapeutic polymer antibody response.
When co-administered with an immunosuppressive drug dose compared to administration of a therapeutic polymer when the concomitant dose is not co-administered with an immunosuppressive drug dose and in the presence of an anti-therapeutic polymer antibody response The method according to a protocol that has been demonstrated to provide a pharmacodynamic effect with a reduced pharmacodynamically effective dose of therapeutic polymer. The method of claim 1, wherein the method further comprises determining a protocol. The method of claim 1 or 2, wherein the method further comprises determining a reduced pharmacodynamically effective dose. The method of any one of claims 1-3, wherein the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration. The method according to any one of claims 1 to 4, wherein the administration is intravenous, intraperitoneal or subcutaneous administration. Providing an immunosuppressant dose, where the immunosuppressant dose is attached to a synthetic nanocarrier; and
A method comprising administering a reduced pharmacodynamically effective dose of a therapeutic polymer in combination with an immunosuppressant dose.
A reduced pharmacodynamically effective dose of the therapeutic polymer is administered in the presence of (A) an anti-therapeutic polymer antibody response and (B) not in combination with an immunosuppressive dose of the therapeutic polymer. The method described above, which is less than the pharmacodynamically effective dose. The method of claim 6, wherein the method further comprises determining a reduced pharmacodynamically effective dose. The method of claim 6 or 7, wherein the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration. The method according to any one of claims 6 to 8, wherein the administration is intravenous, intraperitoneal or subcutaneous administration. Providing an immunosuppressant dose, where the immunosuppressant dose is attached to a synthetic nanocarrier; and
A method comprising administering a pharmacodynamically effective dose of a therapeutic polymer in combination with an immunosuppressive dose to a subject in which an anti-therapeutic polymer antibody response is expected to occur.
The combination administration may improve the pharmacodynamic effect of the therapeutic polymer when used in combination with the immunosuppressant dose as compared to the administration of the therapeutic polymer when it is not administered in combination with the immunosuppressant dose. The method, each of which is in the presence of an anti-therapeutic polymeric antibody response, according to a proven protocol. 10. The method of claim 10, wherein the method further comprises determining a protocol. The method of claim 10 or 11, wherein the method further comprises determining a pharmacodynamically effective dose. The method of any one of claims 10-12, wherein the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration. The method according to any one of claims 10 to 13, wherein the administration is intravenous, intraperitoneal or subcutaneous administration. Providing an immunosuppressant dose, where the immunosuppressant dose is attached to a synthetic nanocarrier;
Administering a pharmacodynamically effective dose of therapeutic macromolecules in combination with an immunosuppressive drug dose to subjects who are expected to experience an anti-therapeutic macromolecule antibody response;
A method comprising recording the improved pharmacodynamic effects following concomitant administration. 15. The method of claim 15, wherein the method further comprises determining a pharmacodynamically effective dose. The method of claim 15 or 16, wherein the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration. The method according to any one of claims 15 to 17, wherein the administration is intravenous, intraperitoneal or subcutaneous administration. To provide therapeutic macromolecules that cause or are expected to cause anti-therapeutic macromolecular antibodies upon repeated dosing in one or more subjects;
Providing an immunosuppressant dose, where the immunosuppressant dose is attached to a synthetic nanocarrier; and
A method comprising administering a therapeutic macromolecule at the same or less dose to a subject repeatedly in combination with an immunosuppressant dose. 19. The method of claim 19, wherein the combination administration follows a protocol that has been demonstrated to result in the maintenance of the pharmacodynamic effects of the therapeutic macromolecule over two or three or more doses of the therapeutic macromolecule to a subject. .. 20. The method of claim 20, wherein the method further comprises determining a protocol. The method of any one of claims 19-21, wherein the method further comprises assessing the pharmacodynamic effect in the subject before and / or after administration of two or three or more doses. The method according to any one of claims 19 to 22, wherein the administration is intravenous, intraperitoneal or subcutaneous administration. Immunosuppressant doses, where immunosuppressant doses are attached to synthetic nanocarriers; and
A composition or kit comprising a reduced pharmacodynamically effective dose of a Therapeutic macromolecule. 24. The composition or kit of claim 24, wherein the composition or kit is for use in any one of the methods provided herein. The composition or kit according to claim 24 or 25, wherein the composition or kit further comprises a pharmaceutically acceptable carrier. Reduced pharmacodynamics of therapeutic macromolecules for use in any one of the methods provided herein, in combination with the immunosuppressant dose, where the immunosuppressant is attached to a synthetic nanocarrier. A composition or kit containing a therapeutically effective dose. 27. The composition or kit of claim 27, further comprising a pharmaceutically acceptable carrier. The method or composition or kit according to any one of claims 1-28, wherein the therapeutic polymer is not attached to the synthetic nanocarrier. The method or composition or kit according to any one of claims 1-28, wherein the therapeutic polymer is attached to a synthetic nanocarrier. The method or composition or kit according to any one of claims 1 to 30, wherein the synthetic nanocarrier does not contain an APC-presentable antigen of a therapeutic polymer. The composition or kit according to any one of claims 24-31, wherein the immunosuppressant dose and the therapeutic macromolecule are contained in separate containers. The composition or kit according to any one of claims 24-31, wherein the immunosuppressant dose and the therapeutic macromolecule are contained in the same container. A reduced pharmacodynamically effective dose of the therapeutic polymer is administered in the presence of (A) an anti-therapeutic polymer antibody response and (B) not in combination with an immunosuppressive dose of the therapeutic polymer. The composition or kit according to any one of claims 24-33 or the method according to claims 1-9, which is at least 30% less than the pharmacodynamically effective dose. The method or composition or kit of claim 34, wherein the reduced pharmacodynamically effective dose is at least 40% less. The method or composition or kit of claim 35, wherein the reduced pharmacodynamically effective dose is at least 50% less. Immunosuppressant doses are statins, mTOR inhibitors, TGF-β signaling agents, corticosteroids, mitochondrial function inhibitors, P38 inhibitors, NF-κβ inhibitors, adenosine receptor agonists, prostaglandin E2 agonists, phosphodiesterase 4 inhibitors, HDACs. The method or composition or kit according to any one of claims 1-36, which comprises an inhibitor or a proteasome inhibitor. The method or composition or kit of claim 37, wherein the mTOR inhibitor is rapamycin. The method or composition or kit according to any one of claims 1-38, wherein the therapeutic macromolecule comprises a therapeutic protein. 39. The method or composition or kit of claim 39, wherein the therapeutic protein is for protein replacement or protein augmentation therapy. Therapeutic polymers are associated with instillable or injectable therapeutic proteins, enzymes, enzyme cofactors, hormones, blood or blood coagulation factors, cytokines, interferons, growth factors, monoclonal antibodies, polyclonal antibodies, or Pompe's disease. The method or composition or kit according to any one of claims 1 to 40, which comprises the protein to be used. Therapeutic proteins that can be infused or injected are tocilizumab, alpha-1 antitrypsin, hematide, alfa interferon alfa-2b, lucin, tesamorelin, ocrelizumab, belimumab, pegloticase, tarigulcerase alfa, agarcidase alfa or velaglucerase alfa. The method or composition or kit of claim 41, comprising celase alpha. 41. The method or composition or kit of claim 41, wherein the enzyme comprises an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase or a ligase. The method or composition or kit of claim 41, wherein the enzyme comprises an enzyme for enzyme replacement therapy for impaired lysosomal accumulation. Enzyme replacement therapy enzymes for lysosomal accumulation disorders are imiglucerase, a-galactosidase A (a-galA), agarsidase beta, acidic α-glucosidase (GAA), alglucosidase alfa, LUMIZYME, MYOZYME, arylsulfase B, laronidase. , ALDURAZYME, Idursulfase, ELAPRASE, arylsulfase B or NAGLAZYME, the method or composition or kit of claim 44. The method or composition or kit of claim 41, wherein the enzyme comprises KRYSTEXXA (pegloticase). The method or composition or kit of claim 41, wherein the monoclonal antibody comprises HUMIRA (adalimumab). The method or composition or kit of claim 41, wherein the cytokine comprises lymphokines, interleukins, chemokines, type 1 or type 2 cytokines. Blood or blood coagulation factors are factor I, factor II, tissue factor, factor V, factor VII, factor VIII, factor IX, factor X, factor Xa, factor XII, factor XIII, Von Villebrand factor, precalicrane, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z related protease inhibitor (ZPI), plasminogen, alpha 2-anti Claims comprising plasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagrant or epoetin alfa. 41. The method or composition or kit according to 4. The method or composition or kit of claim 49, wherein the blood or blood coagulation factor is a factor VIII. The method according to any one of claims 1 to 50, wherein the load of the immunosuppressant attached to the synthetic nanocarrier is between 0.1% and 50% on average over the synthetic nanocarrier. Composition or kit. The method or composition or kit of claim 51, wherein the payload is between 0.1% and 20%. Any of claims 1-52, wherein the synthetic nanocarrier comprises lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, bucky balls, nanowires, virus-like particles, or peptide or protein particles. Or the method or composition or kit according to one paragraph. The method or composition or kit of claim 53, wherein the synthetic nanocarrier comprises lipid nanoparticles. The method or composition or kit of claim 53, wherein the synthetic nanocarrier comprises liposomes. The method or composition or kit of claim 53, wherein the synthetic nanocarrier comprises metal nanoparticles. The method or composition or kit of claim 56, wherein the metal nanoparticles comprise gold nanoparticles. The method or composition or kit of claim 53, wherein the synthetic nanocarrier comprises polymer nanoparticles. 58. The method or composition or kit of claim 58, wherein the polymer nanoparticles comprise a polymer that is a non-methoxy-terminated pluronic polymer. The method or composition or kit according to claim 58 or 59, wherein the polymer nanoparticles comprise polyester, polyester attached to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine. The method or composition or kit of claim 60, wherein the polyester comprises poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid) or polycaprolactone. The method or composition or kit of claim 60 or 61, wherein the polymer nanoparticles comprise a polyester and a polyester attached to a polyether. The method or composition or kit according to any one of claims 60 to 62, wherein the polyether comprises polyethylene glycol or polypropylene glycol. The method or composition or kit according to any one of claims 1 to 63, wherein the average value of the particle size distribution obtained by using the dynamic light scattering of the synthetic nanocarrier is a diameter larger than 100 nm. The method or composition or kit of claim 64, wherein the diameter is greater than 150 nm. 65. The method or composition or kit of claim 65, wherein the diameter is greater than 200 nm. The method or composition or kit of claim 66, wherein the diameter is greater than 250 nm. The method or composition or kit of claim 67, wherein the diameter is greater than 300 nm. Claim 1 in which the aspect ratio of the synthetic nanocarrier is greater than 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7 or 1:10. The method or composition or kit according to any one of ~ 68.

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